Compositions and methods for detecting hepatitis B virus

ABSTRACT

Compositions, methods and kits for detecting viral nucleic acids. Targets that can be detected in accordance with the invention include HBV and/or HIV-1 and/or HCV nucleic acids. Particularly described are oligonucleotides that are useful as hybridization probes and amplification primers that facilitate detection of very low levels of HBV nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/872,538, now U.S. Pat. No. 7,785,844, which is a continuation of U.S.patent application Ser. No. 10/461,790, filed Jun. 13, 2003, abandoned,which claims the benefit of U.S. Provisional Application No. 60/389,393,filed Jun. 14, 2002. The entire disclosures of these prior applicationsare hereby incorporated by reference.

GOVERNMENT INTEREST IN THE INVENTION

Certain aspects of the invention disclosed herein were made withgovernment support under contracts N01-HB-67130 and N01-HB-07148 withthe National Heart, Lung and Blood Institute of the National Institutesof Health. The United States government has certain rights in theseaspects of the invention.

FIELD OF THE INVENTION

The present invention relates to the field of biotechnology. Morespecifically, the invention relates to diagnostic assays for detectingthe nucleic acids of any or all of hepatitis B virus, humanimmunodeficiency virus type 1, and hepatitis C virus.

BACKGROUND OF THE INVENTION

Infection by the hepatitis B virus (HBV) occurs worldwide and is animportant cause of both acute and chronic viral hepatitis. HBV is apartially double-stranded circular DNA virus having a viral particlesize of 42 nm. This particle includes an outer lipoprotein coat and thehepatitis B surface antigen (HBsAg). The HBsAg, circulates in the bloodas a viral particle-bound form, or as a free, noninfectious proteinaggregated into 22-nm spherical and tubular particles. Transmission ofHBV is mediated primarily through blood and/or sexual contact. Theincubation period for this virus ranges as high as 180 days (Gitlin,Clin. Chem. 43:1500 (1997)).

The HBsAg, which can be detected from 2 to 12 weeks after infection withHBV, is the first serologic marker of HBV infection. The presence ofthis marker often precedes symptoms or abnormalities of hepaticbiochemistry by 6-8 weeks. Antibodies specific for the hepatitis B coreantigen, which is contained within the viral particle, usually appear 2weeks after the detection of HBsAg, and remain detectable for up to 6months after the onset of the acute hepatitis (Gitlin, Supra).

The risk of hepatitis virus transmission from transfusions has declineddramatically since post-transfusion hepatitis (PTH) was first recognizedin the 1940s. For example, in 1970 scientists at the NIH reported theresults of a prospective study to determine the incidence of icteric andanicteric hepatitis in patients that had undergone open-heart surgeryand received blood from commercial or volunteer blood donors. Ictericand anicteric hepatitis developed in 51% of the recipients of commercialblood, but in none of the patients who received blood from volunteerdonors.

It was also revealed in 1970 at an NIH Conference that the “Australia”antigen (now known as the hepatitis B surface antigen) was part of aninfectious agent, presumably a hepatitis virus. Only a couple of yearslater, the simultaneous exclusion of commercial blood and HBsAg-positivedonors reduced the incidence of PTH to about 7% of the prior rate(Tobler et al., Clin. Chem. 43:1487 (1997)). These measures dramaticallyimproved the safety of the donated blood supply.

Nucleic acid testing has more recently been undertaken to increase assaysensitivity, thereby ensuring an even higher level of safety for thesupply of donated blood. Assays and reagents for detecting HBV have beenpreviously disclosed in, for example, U.S. Pat. Nos. 5,780,219 and4,562,159; and in published International Patent Application Nos.WO94/08032 and WO95/02690.

SUMMARY OF THE INVENTION

A first aspect of the invention relates to a capture oligonucleotidecomposition that includes a polynucleotide that has a length of up to100 nucleotides, and includes an HBV-complementary sequence that iseither 20 contiguous nucleotides of SEQ ID NO:68, or 25 contiguousnucleotides of SEQ ID NO:69. Further, the polynucleotide is immobilizedto a solid support. In one embodiment of the invention, the solidsupport is a paramagnetic particle. In another embodiment, thepolynucleotide further includes a first homopolymeric sequence, thesolid support is a paramagnetic particle covalently linked to a secondhomopolymeric sequence, and the polynucleotide is immobilized to thesolid support by complementary base pairing between the firsthomopolymeric sequence and the second homopolymeric sequence. In stillanother embodiment, the HBV-complementary sequence of the polynucleotideis any of SEQ ID NOs:70-76. More preferably, the HBV-complementarysequence of the polynucleotide is SEQ ID NO:73. In still anotherembodiment, the HBV-complementary sequence of the polynucleotide is anyof SEQ ID NOs:77-87. More preferably, the HBV-complementary sequence ofthe polynucleotide is SEQ ID NOs:80 or 87.

A second aspect of the invention relates to a kit for detecting HBVtarget nucleic acids. In this instance the invented kit includes atleast one capture oligonucleotide of up to 100 nucleotides in length andincluding an HBV-complementary sequence of at least 20 contiguousnucleotides of SEQ ID NO:68 or a sequence of at least 25 contiguousnucleotides of SEQ ID NO:69. This capture oligonucleotide is immobilizedto a solid support. The kit further includes at least one first strandprimer that includes a downstream HBV-complementary sequence, andoptionally a first strand primer upstream sequence that is notcomplementary to HBV nucleic acids. The downstream HBV-complementarysequence of first strand primer consists of 20-50 contiguous basescontained within SEQ ID NO:2, allowing for the presence of RNA and DNAequivalents and nucleotide analogs. Still further, the kit includes atleast one second strand primer that includes a downstreamHBV-complementary sequence, and optionally a second strand primerupstream sequence that is not complementary to HBV nucleic acids. Thedownstream HBV-complementary sequence of the second strand primerconsists of 20-54 contiguous bases contained within the sequence of SEQID NO:4, allowing for the presence of RNA and DNA equivalents andnucleotide analogs. In one embodiment of the invention, the downstreamHBV-complementary sequence of the at least one first strand primer canbe any of SEQ ID NOs:22-28. More preferably, the at least one captureoligonucleotide is any of SEQ ID NO:73, SEQ ID NO:80 and SEQ ID NO:87.More generally, and with respect to primers contained in the kit, thefirst strand primer upstream sequence that is not complementary to HBVnucleic acids can be a promoter sequence for an RNA polymerase. Inanother embodiment, the downstream HBV-complementary sequence of the atleast one first strand primer can be any of SEQ ID NOs:22-28. In stillanother embodiment, the downstream HBV-complementary sequence of the atleast one second strand primer is any of SEQ ID NOs:5-15. When thedownstream HBV-complementary sequence of the at least one first strandprimer is any of SEQ ID NOs:22-28, the downstream HBV-complementarysequence of the at least one second strand primer is preferably any ofSEQ ID NOs:5-15. In an alternative embodiment, when the downstreamHBV-complementary sequence of the at least one first strand primer isany of SEQ ID NOs:22-28, the downstream HBV-complementary sequence ofthe at least one second strand primer is preferably any of SEQ IDNOs:5-15. According to a different embodiment, the invented kit furtherincludes at least one detectably labeled probe that hybridizes to anamplicon produced in an amplification reaction using the invented firstand second strand primers. Preferred probes have lengths of up to 23nucleotides, and sequences of at least 17 contiguous nucleotidescontained in the sequence of SEQ ID NO:95 or the complement thereof,allowing for the presence of RNA and DNA equivalents and nucleotideanalogs. More preferably, the detectably labeled probe is any of SEQ IDNOs:50-66. In an alternative embodiment, when the downstreamHBV-complementary sequence of the first strand primer is any of SEQ IDNOs:22-28, and when the downstream HBV-complementary sequence of thesecond strand primer is any of SEQ ID NOs:5-15, there is furtherincluded at least one detectably labeled probe having a sequence that isany of SEQ ID NOs:50-66. When the invented kit includes at least onedetectably labeled probe that hybridizes to an amplicon produced in anamplification reaction using the invented first and second strandprimers, the probe can be a molecular beacon that includes atarget-complementary loop sequence of nucleotides. Thistarget-complementary loop sequence of nucleotides is preferably asequence of 12-20 contiguous nucleotides contained within the sequenceof SEQ ID NO:126. More preferably, the target-complementary loopsequence of nucleotides is any of SEQ ID NOs:127-133. Generallyspeaking, the at least one second strand primer of the invented kit mayinclude two second strand primers. When this is the case, the at leastone first strand primer preferably is any of SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:26 and SEQ ID NO:27. Generally speaking, the at least onefirst strand primer of the invented kit may include two first strandprimers, and the at least one second strand primer may include twosecond strand primers. In a highly preferred embodiment of this kit,there is further included an HBV pseudo target that comprises RNA.

A third aspect of the invention also relates to a kit for amplifying HBVtarget nucleic acids that may be present in a biological sample. Thiskit includes a first strand primer that includes a downstreamHBV-complementary sequence, and optionally a first strand primerupstream sequence that is not complementary to HBV nucleic acids. Thedownstream HBV-complementary sequence of the first strand primerconsists of 20-50 contiguous bases contained within SEQ ID NO:2,allowing for the presence of RNA and DNA equivalents and nucleotideanalogs. The kit also contains a second strand primer that includes adownstream HBV-complementary sequence, and optionally a second strandprimer upstream sequence that is not complementary to HBV nucleic acids.The downstream HBV-complementary sequence of the second strand primerconsists of 20-54 contiguous bases contained within the sequence of SEQID NO:4, allowing for the presence of RNA and DNA equivalents andnucleotide analogs. In a particular embodiment of the invention, thefirst strand primer upstream sequence is a promoter sequence for an RNApolymerase. In certain other embodiments, the downstreamHBV-complementary sequence of the first strand primer consists of 20-24contiguous bases contained within SEQ ID NO:2. In a more preferredembodiment, the downstream HBV-complementary sequence of the firststrand primer is any of SEQ ID NOs:22-28, or still more preferably anyof SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:26 or SEQ ID NO:27. In certainspecific embodiments of the invention, the downstream HBV-complementarysequence of the second strand primer is any of SEQ ID NOs:5-15, or stillmore preferably either SEQ ID NO:11 or SEQ ID NO:15. In accordance withanother embodiment, when the downstream HBV-complementary sequence ofthe first strand primer is any of SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:26 or SEQ ID NO:27, the downstream HBV-complementary sequence of thesecond strand primer is preferably any of SEQ ID NOs:5-15. When this isthe case, the downstream HBV-complementary sequence of the second strandprimer is preferably either SEQ ID NO:11 and SEQ ID NO:15. In a highlypreferred embodiment of this invention, the kit further includes atleast one detectably labeled probe that hybridizes to an HBV ampliconwhich is produced in an amplification reaction carried out using thefirst strand primer and the second strand primer. In a still more highlypreferred embodiment, the detectably labeled probe includes the sequenceof SEQ ID NO:50 or the complement thereof, SEQ ID NO:57 or thecomplement thereof, or SEQ ID NO:55 or the complement of thereof. In analternative embodiment, the detectably labeled probe is a molecularbeacon that includes a target-complementary loop sequence ofnucleotides. This target-complementary loop sequence of nucleotidestypically consists of 12-20 contiguous nucleotides contained in thesequence of SEQ ID NO:126 or the complement thereof, allowing for thepresence of nucleotide analogs and RNA and DNA equivalent bases. In adifferent embodiment, the kit includes an additional first strandprimer. In a preferred embodiment, when the kit includes an additionalfirst strand primer, the downstream HBV-complementary sequence of thesecond strand primer has the sequence of SEQ ID NO:11 or SEQ ID NO:15.In still a different embodiment, the kit further includes an additionalsecond strand primer. For example, when the kit includes an additionalsecond strand primer, the downstream HBV-complementary sequence of thefirst strand primer is preferably any of SEQ ID NO:22, SEQ ID NO:23, SEQID NO:26 or SEQ ID NO:27. In still a different embodiment, the kitfurther includes both an additional first strand primer and anadditional second strand primer. This embodiment is illustrated by a kitwherein the downstream HBV-complementary sequence of the first strandprimer includes SEQ ID NO:22, wherein the additional first strand primerincludes SEQ ID NO:23, wherein the downstream HBV-complementary sequenceof the second strand primer includes SEQ ID NO:15, and wherein theadditional second strand primer includes SEQ ID NO:11. In a highlypreferred embodiment of this kit, there is further included an HBVpseudo target that is made of RNA. In another embodiment, when the kitfurther includes both an additional first strand primer and anadditional second strand primer, the downstream HBV-complementarysequence of the first strand primer can include SEQ ID NO:23, theadditional first strand primer can include SEQ ID NO:26, the downstreamHBV-complementary sequence of the second strand primer can include SEQID NO:15, and the additional second strand primer can include SEQ IDNO:11. In another embodiment, when the kit further includes both anadditional first strand primer and an additional second strand primer,the downstream HBV-complementary sequence of the first strand primer caninclude SEQ ID NO:24, the additional first strand primer can include SEQID NO:17, the second strand primer can include SEQ ID NO:15, and theadditional second strand primer can include SEQ ID NO:5. In anotherembodiment, when the kit further includes both an additional firststrand primer and an additional second strand primer, the first strandprimer can include SEQ ID NO:24, the additional first strand primer caninclude SEQ ID NO:20, the second strand primer can include SEQ ID NO:15,and the additional second strand primer can include SEQ ID NO:5.Generally speaking, kits in accordance with the invention can furtherinclude either or both of primers for amplifying an HIV-1 target nucleicacid, and primers for amplifying an HCV target nucleic acid.

DEFINITIONS

The following terms have the following meanings for the purposes of thisdisclosure, unless expressly stated to the contrary herein.

As used herein, a “biological sample” is any tissue orpolynucleotide-containing material obtained from a human. Biologicalsamples in accordance with the invention include peripheral blood,plasma, serum, bone marrow, biopsy tissue including lymph nodes,respiratory tissue or exudates, gastrointestinal tissue, cervical swabsamples, semen or other body fluids, tissues or materials. A biologicalsample may be treated to disrupt tissue or cell structure, therebyreleasing intracellular components into a solution which may containenzymes, buffers, salts, detergents and the like.

As used herein, “polynucleotide” means either RNA or DNA, along with anysynthetic nucleotide analogs or other molecules that may be present inthe sequence and that do not prevent hybridization of the polynucleotidewith a second molecule having a complementary sequence. The termincludes polymers containing analogs of naturally occurring nucleotidesand particularly includes analogs having a methoxy group at the 2′position of the ribose (OMe).

As used herein, a “detectable label” is a chemical species that can bedetected or can lead to a detectable response. Detectable labels inaccordance with the invention can be linked to polynucleotide probeseither directly or indirectly, and include radioisotopes, enzymes,haptens, chromophores such as dyes or particles that impart a detectablecolor (e.g., latex beads or metal particles), luminescent compounds(e.g., bioluminescent, phosphorescent or chemiluminescent moieties) andfluorescent compounds.

A “homogeneous detectable label” refers to a label that can be detectedin a homogeneous fashion by determining whether the label is on a probehybridized to a target sequence. That is, homogeneous detectable labelscan be detected without physically removing hybridized from unhybridizedforms of the label or labeled probe. These labels have been described indetail by Arnold et al., U.S. Pat. No. 5,283,174; Woodhead et al., U.S.Pat. No. 5,656,207; and Nelson et al., U.S. Pat. No. 5,658,737.Preferred labels for use in homogenous assays include chemiluminescentcompounds (e.g., see Woodhead et al., U.S. Pat. No. 5,656,207; Nelson etal., U.S. Pat. No. 5,658,737; and Arnold, Jr., et al., U.S. Pat. No.5,639,604). Preferred chemiluminescent labels are acridinium ester(“AE”) compounds, such as standard AE or derivatives thereof (e.g.,naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2,7-dimethyl-AE,4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE,ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE,ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE, 1- or3-methyl-meta-difluoro-AE, and 2-methyl-AE).

As used herein, “amplification” refers to an in vitro procedure forobtaining multiple copies of a target nucleic acid sequence, itscomplement or fragments thereof.

By “target nucleic acid” or “target” is meant a nucleic acid containinga target nucleic acid sequence.

By “target nucleic acid sequence,” “target nucleotide sequence,” “targetsequence” or “target region” is meant a specific deoxyribonucleotide orribonucleotide sequence comprising all or part of the nucleotidesequence of a single-stranded nucleic acid molecule, and thedeoxyribonucleotide or ribonucleotide sequence complementary thereto.

By “transcription associated amplification” is meant any type of nucleicacid amplification that uses an RNA polymerase to produce multiple RNAtranscripts from a nucleic acid template. One example of a transcriptionassociated amplification method, called “Transcription MediatedAmplification” (TMA), generally employs an RNA polymerase, a DNApolymerase, deoxyribonucleoside triphosphates, ribonucleosidetriphosphates, and a promoter-template complementary oligonucleotide,and optionally may include one or more analogous oligonucleotides.Variations of TMA are well known in the art as disclosed in detail inBurg et al., U.S. Pat. No. 5,437,990; Kacian et al., U.S. Pat. Nos.5,399,491 and 5,554,516; Kacian et al., PCT No. WO 93/22461; Gingeras etal., PCT No. WO 88/01302; Gingeras et al., PCT No. WO 88/10315; Malek etal., U.S. Pat. No. 5,130,238; Urdea et al., U.S. Pat. Nos. 4,868,105 and5,124,246; McDonough et al., PCT No. WO 94/03472; and Ryder et al., PCTNo. WO 95/03430. The methods of Kacian et al. are preferred forconducting nucleic acid amplification procedures of the type disclosedherein.

As used herein, an “oligonucleotide” or “oligomer” is a polymeric chainof at least two, generally between about five and about 100, chemicalsubunits, each subunit comprising a nucleotide base moiety, a sugarmoiety, and a linking moiety that joins the subunits in a linear spatialconfiguration. Common nucleotide base moieties are guanine (G), adenine(A), cytosine (C), thymine (T) and uracil (U), although other rare ormodified nucleotide bases able to hydrogen bond are well known to thoseskilled in the art. Oligonucleotides may optionally include analogs ofany of the sugar moieties, the base moieties, and the backboneconstituents. Preferred oligonucleotides of the present invention fallin a size range of about 10 to about 60 residues. Oligonucleotides maybe purified from naturally occurring sources, but preferably aresynthesized using any of a variety of well known enzymatic or chemicalmethods.

As used herein, a “probe” is an oligonucleotide that hybridizesspecifically to a target sequence in a nucleic acid, preferably in anamplified nucleic acid, under conditions that promote hybridization, toform a detectable hybrid. A probe may contain a detectable moiety whicheither may be attached to the end(s) of the probe or may be internal.The nucleotides of the probe which combine with the targetpolynucleotide need not be strictly contiguous, as may be the case witha detectable moiety internal to the sequence of the probe. Detection mayeither be direct (i.e., resulting from a probe hybridizing directly tothe target sequence or amplified nucleic acid) or indirect (i.e.,resulting from a probe hybridizing to an intermediate molecularstructure that links the probe to the target sequence or amplifiednucleic acid). The “target” of a probe generally refers to a sequencecontained within an amplified nucleic acid sequence which hybridizesspecifically to at least a portion of a probe oligonucleotide usingstandard hydrogen bonding (i.e., base pairing). A probe may comprisetarget-specific sequences and other sequences that contribute tothree-dimensional conformation of the probe (e.g., as described inLizardi et al., U.S. Pat. Nos. 5,118,801 and 5,312,728). Sequences thatare “sufficiently complementary” allow stable hybridization of a probeoligonucleotide to a target sequence that is not completelycomplementary to the probe's target-specific sequence.

As used herein, an “amplification primer” is an oligonucleotide thathybridizes to a target nucleic acid, or its complement, and participatesin a nucleic acid amplification reaction. Amplification primers, or moresimply “primers,” may be an optionally modified oligonucleotide which iscapable of hybridizing to a template nucleic acid and which has a 3′ endthat can be extended by a DNA polymerase activity.

By “substantially homologous,” “substantially corresponding” or“substantially corresponds” is meant that the subject oligonucleotidehas a base sequence containing an at least 10 contiguous base regionthat is at least 70% homologous, preferably at least 80% homologous,more preferably at least 90% homologous, and most preferably 100%homologous to an at least 10 contiguous base region present in areference base sequence (excluding RNA and DNA equivalents). Thoseskilled in the art will readily appreciate modifications that could bemade to the hybridization assay conditions at various percentages ofhomology to permit hybridization of the oligonucleotide to the targetsequence while preventing unacceptable levels of non-specifichybridization. The degree of similarity is determined by comparing theorder of nucleobases making up the two sequences and does not take intoconsideration other structural differences which may exist between thetwo sequences, provided the structural differences do not preventhydrogen bonding with complementary bases. The degree of homologybetween two sequences can also be expressed in terms of the number ofbase mismatches present in each set of at least 10 contiguous basesbeing compared, which may range from 0-2 base differences.

By “substantially complementary” is meant that the subjectoligonucleotide has a base sequence containing an at least 10 contiguousbase region that is at least 70% complementary, preferably at least 80%complementary, more preferably at least 90% complementary, and mostpreferably 100% complementary to an at least 10 contiguous base regionpresent in a target nucleic acid sequence (excluding RNA and DNAequivalents). (Those skilled in the art will readily appreciatemodifications that could be made to the hybridization assay conditionsat various percentages of complementarity to permit hybridization of theoligonucleotide to the target sequence while preventing unacceptablelevels of non-specific hybridization.) The degree of complementarity isdetermined by comparing the order of nucleobases making up the twosequences and does not take into consideration other structuraldifferences which may exist between the two sequences, provided thestructural differences do not prevent hydrogen bonding withcomplementary bases. The degree of complementarity between two sequencescan also be expressed in terms of the number of base mismatches presentin each set of at least 10 contiguous bases being compared, which mayrange from 0-2 base mismatches.

By “sufficiently complementary” is meant a contiguous nucleic acid basesequence that is capable of hybridizing to another base sequence byhydrogen bonding between a series of complementary bases. Complementarybase sequences may be complementary at each position in the basesequence of an oligonucleotide using standard base pairing (e.g., G:C,A:T or A:U pairing) or may contain one or more residues that are notcomplementary using standard hydrogen bonding (including abasic“nucleotides”), but in which the entire complementary base sequence iscapable of specifically hybridizing with another base sequence inappropriate hybridization conditions. Contiguous bases are preferably atleast about 80%, more preferably at least about 90%, and most preferablyabout 100% complementary to a sequence to which an oligonucleotide isintended to specifically hybridize. Appropriate hybridization conditionsare well known to those skilled in the art, can be predicted readilybased on base sequence composition, or can be determined empirically byusing routine testing (e.g., See Sambrook et al., Molecular Cloning, ALaboratory Manual, 2^(nd) ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) at §§1.90-1.91, 7.37-7.57, 9.47-9.51 and11.47-11.57 particularly at §§9.50-9.51, 11.12-11.13, 11.45-11.47 and11.55-11.57).

By “capture oligonucleotide” is meant at least one nucleic acidoligonucleotide that provides means for specifically joining a targetsequence and an immobilized oligonucleotide due to base pairhybridization. A capture oligonucleotide preferably includes two bindingregions: a target sequence-binding region and an immobilizedprobe-binding region, usually contiguous on the same oligonucleotide,although the capture oligonucleotide may include a targetsequence-binding region and an immobilized probe-binding region whichare present on two different oligonucleotides joined together by one ormore linkers. For example, an immobilized probe-binding region may bepresent on a first oligonucleotide, the target sequence-binding regionmay be present on a second oligonucleotide, and the two differentoligonucleotides are joined by hydrogen bonding with a linker that is athird oligonucleotide containing sequences that hybridize specificallyto the sequences of the first and second oligonucleotides.

By “immobilized probe” or “immobilized nucleic acid” is meant a nucleicacid that joins, directly or indirectly, a capture oligonucleotide to animmobilized support. An immobilized probe is an oligonucleotide joinedto a solid support that facilitates separation of bound target sequencefrom unbound material in a sample.

By “separating” or “purifying” is meant that one or more components ofthe biological sample are removed from one or more other components ofthe sample. Sample components include nucleic acids in a generallyaqueous solution phase which may also include materials such asproteins, carbohydrates, lipids and labeled probes. Preferably, theseparating or purifying step removes at least about 70%, more preferablyat least about 90% and, even more preferably, at least about 95% of theother components present in the sample.

By “RNA and DNA equivalents” or “RNA and DNA equivalent bases” is meantRNA and DNA molecules having the same complementary base pairhybridization properties. RNA and DNA equivalents have different sugarmoieties (i.e., ribose versus deoxyribose) and may differ by thepresence of uracil in RNA and thymine in DNA. The differences betweenRNA and DNA equivalents do not contribute to differences in homologybecause the equivalents have the same degree of complementarity to aparticular sequence.

By “consisting essentially of” is meant that additional component(s),composition(s) or method step(s) that do not materially change the basicand novel characteristics of the present invention may be included inthe compositions or kits or methods of the present invention. Suchcharacteristics include the ability to selectively detect HBV nucleicacids in biological samples such as whole blood or plasma, at about 100copies of the HBV nucleic acid. Any component(s), composition(s), ormethod step(s) that have a material effect on the basic and novelcharacteristics of the present invention would fall outside of thisterm.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating the various polynucleotidesthat can be used for detecting a target region within the HBV nucleicacid (represented by a thick horizontal line). Positions of thefollowing nucleic acids are shown relative to the target region:“Capture Oligonucleotide” refers to the nucleic acid used to hybridizeto and capture the target nucleic acid prior to amplification, where “T”refers to a tail sequence used to hybridize an immobilizedoligonucleotide having a complementary sequence (not shown); “Non-T7Primer” and “T7 Promoter-Primer” represent two amplification primersused for conducting TMA, where “P” indicates the promoter sequence ofthe T7 promoter-primer; and “Probe” refers to the probe used fordetecting amplified nucleic acid.

FIG. 2 is a line graph showing a quantitative HBV calibrator curve thatemphasizes the linear relationship between input template amount andhybridization signal strength over a broad range of calibrator amounts.

FIG. 3 is a line graph relating the amount of HBV standard input into areal-time nucleic acid amplification reaction (x-axis) and thetime-of-emergence of the measured fluorescent signal above a backgroundthreshold (y-axis).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions, methods and kits forselectively detecting the nucleic acids of hepatitis B virus (HBV)and/or human immunodeficiency virus-1 (HIV-1) and/or hepatitis C virus(HCV). The compositions disclosed herein are useful for amplifying anddetecting these nucleic acids in biological samples such as human blood,serum, plasma or other body fluid or tissue to be tested for thepresence of viral nucleic acids. Many of the amplification primersdisclosed herein advantageously can be used as components of multiplexamplification reactions, wherein several amplicon species can beproduced from a complex assortment of primers and accessorypolynucleotides. For example, the most highly preferred HBV-specificprimers disclosed herein can be used in multiplex amplificationreactions that are capable of amplifying polynucleotides of theunrelated viruses without substantially compromising the sensitivitiesof those assays.

The probes, primers and methods disclosed herein can be used either indiagnostic applications or for screening donated blood and bloodproducts or other tissues that may contain infectious particles.Additionally, there is disclosed a quantitative assay that employsnucleic acid amplification techniques to measure the number of copies ofHBV nucleic acid in a biological sample. This quantitative assayrepresents an important tool for monitoring viral load in patientsundergoing antiviral therapy.

Introduction and Overview

The present invention includes compositions (nucleic acid captureoligonucleotides, amplification oligonucleotides and probes), methodsand kits for detecting HBV nucleic acids in a biological sample. Todesign oligonucleotide sequences appropriate for such uses, known HBVDNA sequences, including subtypes, were first aligned by matchingregions having similar sequences and then comparing the sequences toidentify candidate regions of the viral genome that could serve asreagents in a diagnostic assay. Based on these comparisons, the “Sgene/pol gene” region of the HBV genome was selected for detection usingthe capture oligonucleotides, primers and probes shown schematically inFIG. 1. Portions of sequences containing relatively few variants betweenthe compared sequences were chosen as starting points for designingsynthetic oligonucleotides suitable for use in capture, amplificationand detection of amplified sequences. Other considerations in designingoligonucleotides included the relative GC content of the sequence(ranging from about 30% to about 55%), and the relative absence ofpredicted secondary structure (e.g., hairpin turns formingintramolecular hybrids) within a sequence.

Based on these analyses, the capture oligonucleotide, amplificationprimer and probe sequences presented below were designed. Those havingan ordinary level of skill in the art will appreciate that primersequences specific for HBV, with or without a T7 promoter sequence, maybe used as primers in the various primer-based in vitro amplificationmethods described below. Additionally, it is also contemplated that thehybridization probes disclosed herein could be used as amplificationprimers, and that the amplification primers disclosed herein could beused as hybridization probes. Still further, it is contemplated that thehybridization probes disclosed herein could be used as captureoligonucleotides, and that the capture oligonucleotides disclosed hereincould be used as hybridization probes. Even still further, it iscontemplated that the amplification primers disclosed herein could beused as capture oligonucleotides, and that the capture oligonucleotidesdisclosed herein could be used as amplification primers.

Useful Amplification Methods

Amplification methods useful in connection with the present inventioninclude: Transcription Mediated Amplification (TMA), Nucleic AcidSequence-Based Amplification (NASBA), the Polymerase Chain Reaction(PCR), Strand Displacement Amplification (SDA), and amplificationmethods using self-replicating polynucleotide molecules and replicationenzymes such as MDV-1 RNA and Q-beta enzyme. Methods for carrying outthese various amplification techniques respectively can be found in U.S.Pat. No. 5,399,491, published European patent application EP 0 525 882,U.S. Pat. No. 4,965,188, U.S. Pat. No. 5,455,166, U.S. Pat. No.5,472,840 and Lizardi et al., BioTechnology 6:1197 (1988). Thedisclosures of these documents which describe how to perform nucleicacid amplification reactions are hereby incorporated by reference.

In a highly preferred embodiment of the invention, HBV nucleic acidsequences are amplified using a TMA protocol. According to thisprotocol, the reverse transcriptase which provides the DNA polymeraseactivity also possesses an endogenous RNase H activity. One of theprimers used in this procedure contains a promoter sequence positionedupstream of a sequence that is complementary to one strand of a targetnucleic acid that is to be amplified. In the first step of theamplification, a promoter-primer hybridizes to the HBV target DNA at adefined site. Reverse transcriptase creates a complementary DNA copy ofthe target DNA by extension from the 3′ end of the promoter-primer.Following interaction of an opposite strand primer with the newlysynthesized DNA strand, a second strand of DNA is synthesized from theend of the primer by reverse transcriptase, thereby creating adouble-stranded DNA molecule. RNA polymerase recognizes the promotersequence in this double-stranded DNA template and initiatestranscription. Each of the newly synthesized RNA amplicons re-enters theTMA process and serves as a template for a new round of replication,thereby leading to an exponential expansion of the RNA amplicon. Sinceeach of the DNA templates can make 100-1000 copies of RNA amplicon, thisexpansion can result in the production of 10 billion amplicons in lessthan one hour. The entire process is autocatalytic and is performed at aconstant temperature.

Structural Features of Primers

As indicated above, a “primer” refers to an optionally modifiedoligonucleotide which is capable of hybridizing to a template nucleicacid and which has a 3′ end that can be extended by a DNA polymeraseactivity. The 5′ region of the primer may be non-complementary to thetarget nucleic acid. If the 5′ non-complementary region includes apromoter sequence, it is referred to as a “promoter-primer.” Thoseskilled in the art will appreciate that any oligonucleotide that canfunction as a primer (i.e., an oligonucleotide that hybridizesspecifically to a target sequence and has a 3′ end capable of extensionby a DNA polymerase activity) can be modified to include a 5′ promotersequence, and thus could function as a promoter-primer. Similarly, anypromoter-primer can be modified by removal of, or synthesis without, apromoter sequence and still function as a primer.

Nucleotide base moieties of primers may be modified (e.g., by theaddition of propyne groups), as long as the modified base moiety retainsthe ability to form a non-covalent association with G, A, C, T or U, andas long as an oligonucleotide comprising at least one modifiednucleotide base moiety is not sterically prevented from hybridizing witha single-stranded nucleic acid. As indicated below in connection withthe chemical composition of useful probes, the nitrogenous bases ofprimers in accordance with the invention may be conventional bases (A,G, C, T, U), known analogs thereof (e.g., inosine or “I”; see TheBiochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11^(th) ed.,1992), known derivatives of purine or pyrimidine bases (e.g., N⁴-methyldeoxygaunosine, deaza- or aza-purines and deaza- or aza-pyrimidines,pyrimidine bases having substituent groups at the 5 or 6 position,purine bases having an altered or a replacement substituent at the 2, 6or 8 positions, 2-amino-6-methylaminopurine, O⁶-methylguanine,4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines (see, Cook,PCT Int'l Pub. No. WO 93/13121) and “abasic” residues where the backboneincludes no nitrogenous base for one or more residues of the polymer(see Arnold et al., U.S. Pat. No. 5,585,481). Common sugar moieties thatcomprise the primer backbone include ribose and deoxyribose, although2′-O-methyl ribose (OMe), halogenated sugars, and other modified sugarmoieties may also be used. Usually, the linking group of the primerbackbone is a phosphorus-containing moiety, most commonly aphosphodiester linkage, although other linkages, such as, for example,phosphorothioates, methylphosphonates, and non-phosphorus-containinglinkages such as peptide-like linkages found in “peptide nucleic acids”(PNA) also are intended for use in the assay disclosed herein.

Useful Probe Labeling Systems and Detectable Moieties

Essentially any labeling and detection system that can be used formonitoring specific nucleic acid hybridization can be used inconjunction with the present invention. Included among the collection ofuseful labels are radiolabels, enzymes, haptens, linkedoligonucleotides, chemiluminescent molecules and redox-active moietiesthat are amenable to electronic detection methods. Preferredchemiluminescent molecules include acridinium esters of the typedisclosed by Arnold et al., in U.S. Pat. No. 5,283,174 for use inconnection with homogenous protection assays, and of the type disclosedby Woodhead et al., in U.S. Pat. No. 5,656,207 for use in connectionwith assays that quantify multiple targets in a single reaction. Thedisclosures contained in these patent documents are hereby incorporatedby reference. Preferred electronic labeling and detection approaches aredisclosed in U.S. Pat. Nos. 5,591,578 and 5,770,369, and the publishedinternational patent application WO 98/57158, the disclosures of whichare hereby incorporated by reference. Redox active moieties useful aslabels in the present invention include transition metals such as Cd,Mg, Cu, Co, Pd, Zn; Fe and Ru.

Particularly preferred detectable labels for probes in accordance withthe present invention are detectable in homogeneous assay systems (i.e.,where, in a mixture, bound labeled probe exhibits a detectable change,such as stability or differential degradation, compared to unboundlabeled probe). A preferred label for use in homogenous assays is achemiluminescent compound (e.g., as described by Woodhead et al., inU.S. Pat. No. 5,656,207; by Nelson et al., in U.S. Pat. No. 5,658,737;or by Arnold et al., in U.S. Pat. No. 5,639,604). Particularly preferredchemiluminescent labels include acridinium ester (“AE”) compounds, suchas standard AE or derivatives thereof, such as naphthyl-AE, ortho-AE, 1-or 3-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE,ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE,ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or3-methyl-ortho-fluoro-AE, 1- or 3-methyl-meta-difluoro-AE, and2-methyl-AE.

In some applications, probes exhibiting at least some degree ofself-complementarity are desirable to facilitate detection ofprobe:target duplexes in a test sample without first requiring theremoval of unhybridized probe prior to detection. By way of example,structures referred to as “Molecular Torches” are designed to includedistinct regions of self-complementarity (coined “the target bindingdomain” and “the target closing domain”) which are connected by ajoining region and which hybridize to one another under predeterminedhybridization assay conditions. When exposed to denaturing conditions,the two complementary regions (which may be fully or partiallycomplementary) of the Molecular Torch melt, leaving the target bindingdomain available for hybridization to a target sequence when thepredetermined hybridization assay conditions are restored. MolecularTorches are designed so that the target binding domain favorshybridization to the target sequence over the target closing domain. Thetarget binding domain and the target closing domain of a Molecular Torchinclude interacting labels (e.g., luminescent/quencher) positioned sothat a different signal is produced when the Molecular Torch isself-hybridized as opposed to when the Molecular Torch is hybridized toa target nucleic acid, thereby permitting detection of probe:targetduplexes in a test sample in the presence of unhybridized probe having aviable label associated therewith. Molecular Torches are fully describedin International Publication No. WO 00/01850, the disclosure of which ishereby incorporated by reference.

Another example of a self-complementary hybridization assay probe thatmay be used in conjunction with the invention is a structure commonlyreferred to as a “Molecular Beacon.” Molecular Beacons comprise nucleicacid molecules having a target complement sequence, an affinity pair (ornucleic acid arms) holding the probe in a closed conformation in theabsence of a target nucleic acid sequence, and a label pair thatinteracts when the probe is in a closed conformation. Hybridization ofthe target nucleic acid and the target complement sequence separates themembers of the affinity pair, thereby shifting the probe to an openconfirmation. The shift to the open confirmation is detectable due toreduced interaction of the label pair, which may be, for example, afluorophore and a quencher. Molecular Beacons are described in U.S. Pat.No. 5,925,517, the disclosure of which is hereby incorporated byreference. Molecular beacons useful for detecting HBV-specific nucleicacid sequences may be created by appending to either end of one of theprobe sequences disclosed herein, a first nucleic acid arm comprising afluorophore and a second nucleic acid arm comprising a quencher moiety.In this configuration, the HBV-specific probe sequence disclosed hereinserves as the target-complementary “loop” portion of the resultingmolecular beacon.

Molecular beacons preferably are labeled with an interactive pair ofdetectable labels. Examples of detectable labels that are preferred asmembers of an interactive pair of labels interact with each other byFRET or non-FRET energy transfer mechanisms. Fluorescence resonanceenergy transfer (FRET) involves the radiationless transmission of energyquanta from the site of absorption to the site of its utilization in themolecule, or system of molecules, by resonance interaction betweenchromophores, over distances considerably greater than interatomicdistances, without conversion to thermal energy, and without the donorand acceptor coming into kinetic collision. The “donor” is the moietythat initially absorbs the energy, and the “acceptor” is the moiety towhich the energy is subsequently transferred. In addition to FRET, thereare at least three other “non-FRET” energy transfer processes by whichexcitation energy can be transferred from a donor to an acceptormolecule.

When two labels are held sufficiently close that energy emitted by onelabel can be received or absorbed by the second label, whether by a FRETor non-FRET mechanism, the two labels are said to be in “energy transferrelationship” with each other. This is the case, for example, when amolecular beacon is maintained in the closed state by formation of astem duplex, and fluorescent emission from a fluorophore attached to onearm of the probe is quenched by a quencher moiety on the opposite arm.

Highly preferred label moieties for the invented molecular beaconsinclude a fluorophore and a second moiety having fluorescence quenchingproperties (i.e., a “quencher”). In this embodiment, the characteristicsignal is likely fluorescence of a particular wavelength, butalternatively could be a visible light signal. When fluorescence isinvolved, changes in emission are preferably due to FRET, or toradiative energy transfer or non-FRET modes. When a molecular beaconhaving a pair of interactive labels in the closed state is stimulated byan appropriate frequency of light, a fluorescent signal is generated ata first level, which may be very low. When this same probe is in theopen state and is stimulated by an appropriate frequency of light, thefluorophore and the quencher moieties are sufficiently separated fromeach other that energy transfer between them is substantially precluded.Under that condition, the quencher moiety is unable to quench thefluorescence from the fluorophore moiety. If the fluorophore isstimulated by light energy of an appropriate wavelength, a fluorescentsignal of a second level, higher than the first level, will begenerated. The difference between the two levels of fluorescence isdetectable and measurable. Using fluorophore and quencher moieties inthis manner, the molecular beacon is only “on” in the “open”conformation and indicates that the probe is bound to the target byemanating an easily detectable signal. The conformational state of theprobe alters the signal generated from the probe by regulating theinteraction between the label moieties.

Examples of donor/acceptor label pairs that may be used in connectionwith the invention, making no attempt to distinguish FRET from non-FRETpairs, include fluorescein/tetramethylrhodamine, IAEDANS/fluororescein,EDANS/DABCYL, coumarin/DABCYL, fluorescein/fluorescein, BODIPY FL/BODIPYFL, fluorescein/DABCYL, lucifer yellow/DABCYL, BODIPY/DABCYL,eosine/DABCYL, erythrosine/DABCYL, tetramethylrhodamine/DABCYL, TexasRed/DABCYL, CY5/BH1, CY5/BH2, CY3/BH1, CY3/BH2 and fluorescein/QSY7 dye.Those having an ordinary level of skill in the art will understand thatwhen donor and acceptor dyes are different, energy transfer can bedetected by the appearance of sensitized fluorescence of the acceptor orby quenching of donor fluorescence. When the donor and acceptor speciesare the same, energy can be detected by the resulting fluorescencedepolarization. Non-fluorescent acceptors such as DABCYL and the QSY 7dyes advantageously eliminate the potential problem of backgroundfluorescence resulting from direct (i.e., non-sensitized) acceptorexcitation. Preferred fluorophore moieties that can be used as onemember of a donor-acceptor pair include fluorescein, ROX, and the CYdyes (such as CY5). Highly preferred quencher moieties that can be usedas another member of a donor-acceptor pair include DABCYL and the BLACKHOLE QUENCHER moieties which are available from Biosearch Technologies,Inc., (Novato, Calif.).

Synthetic techniques and methods of bonding labels to nucleic acids anddetecting labels are well known in the art (e.g., see Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 10; Nelson etal., U.S. Pat. No. 5,658,737; Woodhead et al., U.S. Pat. No. 5,656,207;Hogan et al., U.S. Pat. No. 5,547,842; Arnold et al., U.S. Pat. No.5,283,174; Kourilsky et al., U.S. Pat. No. 4,581,333), and Becker etal., European Patent App. No. 0 747 706.

Chemical Composition of Probes

Probes in accordance with the invention comprise polynucleotides orpolynucleotide analogs and may carry a detectable label covalentlybonded thereto. Nucleosides or nucleoside analogs of the probe comprisenitrogenous heterocyclic bases, or base analogs, where the nucleosidesare linked together, for example by phosphohdiester bonds to form apolynucleotide. Accordingly, a probe may comprise conventionalribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA), but also maycomprise chemical analogs of these molecules. The “backbone” of a probemay be made up of a variety of linkages known in the art, including oneor more sugar-phosphodiester linkages, peptide-nucleic acid bonds(sometimes referred to as “peptide nucleic acids” as described byHyldig-Nielsen et al., PCT Intl Pub. No. WO 95/32305), phosphorothioatelinkages, methylphosphonate linkages or combinations thereof. Sugarmoieties of the probe may be either ribose or deoxyribose, or similarcompounds having known substitutions, such as, for example, 2′-O-methylribose and 2′ halide substitutions (e.g., 2′-F). The nitrogenous basesmay be conventional bases (A, G, C, T, U), known analogs thereof (e.g.,inosine or “I”; see The Biochemistry of the Nucleic Acids 5-36, Adams etal., ed., 11^(th) ed., 1992), known derivatives of purine or pyrimidinebases (e.g., N⁴-methyl deoxygaunosine, deaza- or aza-purines and deaza-or aza-pyrimidines, pyrimidine bases having substituent groups at the 5or 6 position, purine bases having an altered or a replacementsubstituent at the 2, 6 or 8 positions, 2-amino-6-methylaminopurine,O⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines (see, Cook,PCT Intl Pub. No. WO 93/13121) and “abasic” residues where the backboneincludes no nitrogenous base for one or more residues of the polymer(see Arnold et al., U.S. Pat. No. 5,585,481). A nucleic acid maycomprise only conventional sugars, bases and linkages found in RNA andDNA, or may include both conventional components and substitutions(e.g., conventional bases linked via a methoxy backbone, or a nucleicacid including conventional bases and one or more base analogs).

Selection of Amplification Primers and Detection Probes Specific for HBV

Useful guidelines for designing amplification primers and probes withdesired characteristics are described herein. The optimal sites foramplifying and probing HBV nucleic acids contain two, and preferablythree, conserved regions greater than about 15 bases in length, withinabout 200 bases, and preferably within 110 bases, of contiguoussequence. The degree of amplification observed with a set of primers orpromoter-primers depends on several factors, including the ability ofthe oligonucleotides to hybridize to their complementary sequences andtheir ability to be extended enzymatically. Because the extent andspecificity of hybridization reactions are affected by a number offactors, manipulation of those factors will determine the exactsensitivity and specificity of a particular oligonucleotide, whetherperfectly complementary to its target or not. The effects of varyingassay conditions are known to those skilled in the art, and aredescribed by Hogan et al., in U.S. Pat. No. 5,840,488, the disclosure ofwhich is hereby incorporated by reference.

The length of the target nucleic acid sequence and, accordingly, thelength of the primer sequence or probe sequence can be important. Insome cases, there may be several sequences from a particular targetregion, varying in location and length, which will yield primers orprobes having the desired hybridization characteristics. While it ispossible for nucleic acids that are not perfectly complementary tohybridize, the longest stretch of perfectly homologous base sequencewill normally primarily determine hybrid stability.

Amplification primers and probes should be positioned to minimize thestability of the oligonucleotide:nontarget (i.e., nucleic acid withsimilar sequence to target nucleic acid) nucleic acid hybrid. It ispreferred that the amplification primers and detection probes are ableto distinguish between target and non-target sequences. In designingprimers and probes, the differences in these Tm values should be aslarge as possible (e.g., at least 2° C. and preferably 5° C.).

Regions of the nucleic acid which are known to form strong internalstructures inhibitory to hybridization are less preferred as primers orprobes. Examples of such structures include hairpin loops. Likewise,oligonucleotides with extensive self-complementarity should be avoided.

The degree of non-specific extension (primer-dimer or non-targetcopying) can also affect amplification efficiency. For this reason,primers are selected to have low self- or cross-complementarity,particularly at the 3′ ends of the sequence. Long homopolymer tracts andhigh GC content are avoided to reduce spurious primer extension.Commercially available computer software can aid in this aspect of thedesign. Available computer programs include MacDNASIS™ 2.0 (HitachiSoftware Engineering American Ltd.) and OLIGO ver. 4.1 (NationalBioscience).

Those having an ordinary level of skill in the art will appreciate thathybridization involves the association of two single strands ofcomplementary nucleic acid to form a hydrogen bonded double strand. Itis implicit that if one of the two strands is wholly or partiallyinvolved in a hybrid, then that strand will be less able to participatein formation of a new hybrid. By designing primers and probes so thatsubstantial portions of the sequences of interest are single stranded,the rate and extent of hybridization may be greatly increased. If thetarget is an integrated genomic sequence, then it will naturally occurin a double stranded form (as is the case with the product of thepolymerase chain reaction). These double-stranded targets are naturallyinhibitory to hybridization with a probe and require denaturation priorto the hybridization step.

The rate at which a polynucleotide hybridizes to its target is a measureof the thermal stability of the target secondary structure in the targetbinding region. The standard measurement of hybridization rate is theC₀t_(1/2) which is measured as moles of nucleotide per liter multipliedby seconds. Thus, it is the concentration of probe multiplied by thetime at which 50% of maximal hybridization occurs at that concentration.This value is determined by hybridizing various amounts ofpolynucleotide to a constant amount of target for a fixed time. TheC₀t_(1/2) is found graphically by standard procedures familiar to thosehaving an ordinary level of skill in the art.

Preferred Amplification Primers

Many of the oligonucleotides, including primers and probes, disclosedherein were derived from the sequence of the HBV, subtype ADW. Theentire nucleotide sequence of the cloned hepatitis B virus DNA, subtypeADW can be accessed through GENBANK Accession No. V00866.

Generally speaking, primers useful for conducting amplificationreactions in accordance with the invention have a downstreamHBV-complementary sequence, and optionally an upstream sequence that isnot complementary to the HBV target. The HBV-complementary sequences ofcertain primers which are complementary to one strand of the HBV targetnucleic acid (i.e., “first strand” primers) preferably are 20-50 basesin length and have 20-50 contiguous bases of SEQ ID NO:2, allowing forRNA and DNA equivalents and for substitution of nucleotide analogs. TheHBV-complementary sequences of certain primers which are complementaryto the second, or opposite strand of the HBV target nucleic acid (i.e.,“second strand” primers) preferably are 20-54 bases in length and have20-54 contiguous bases of SEQ ID NO:4, allowing for RNA and DNAequivalents and for substitution of nucleotide analogs. Of course, theoptional upstream sequences which are not complementary to the HBVtarget will add to the overall lengths of the primers. An example of anoptional upstream sequence would be a promoter for an RNA polymerase.

Primers useful for conducting amplification reactions can have differentlengths. For example, amplification oligonucleotides complementary toone strand of the HBV target nucleic acid sequence (i.e., first strandprimers) preferably have lengths of up to 100 bases, more preferablyfrom 18-60 bases, and have HBV-complementary sequences ranging in lengthfrom 18-27 bases, or more preferably from 20-23 bases and include atleast 18 contiguous bases, allowing for substitution of one or morenitrogenous base analogs, substantially corresponding to the sequencegiven by GTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATATTCCTCTTCAT CCTGCTGCTAT(SEQ ID NO:1). Examples of primers, including promoter-primers, fallingwithin this group include SEQ ID NOs:16-49. Even more highly preferredprimers for amplifying HBV nucleic acids have lengths of up to 100bases, more preferably from 20-60 bases, and have HBV-complementarysequences that include at least 20 contiguous bases, more preferably20-24 contiguous bases, allowing for substitution of one or morenitrogenous base analogs, contained within a sequence substantiallycorresponding to CTGGATGTGTCTGCGGCGTTTTATCATTCCTCTTCATCCTGCTGCTAT (SEQID NO:2). Examples of primers falling within this group include SEQ IDNOs:22-28, and the respective T7 promoter-primers of SEQ ID NOs:39-45.Another highly preferred collection of primers for amplifying HBVnucleic acids have lengths of up to 100 bases, more preferably from20-60 bases, and have HE V-complementary sequences that include at least23 contiguous bases, more preferably 23-24 contiguous bases, allowingfor substitution of one or more nitrogenous base analogs, containedwithin a sequence substantially corresponding toATCATATTCCTCTTCATCCTGCTGCTAT (SEQ ID NO:3). Examples of primers fallingwithin this group include SEQ ID NOs:24-28, and the respective T7promoter-primers of SEQ ID NOs:41-45. When using a TMA reaction toamplify HBV sequences, primers having these characteristics are highlypreferred for use as promoter-primers, such as T7 promoter-primers. Ofcourse, if the primer is a T7 promoter-primer there will be included atthe 5′ end a T7 promoter sequence which typically adds about 27-33 basesto the length of the primer. Examples of promoter-primers includeoligonucleotides having the sequences given by SEQ ID NOs:33-49. Thepromoter-primers disclosed herein are particularly desirable forperforming nucleic acid amplification reactions using theabove-referenced TMA procedure.

Other primers (i.e., second strand primers) that can be used with theabove-described primers in any combination for carrying outamplification reactions are complementary to the opposite strand of theHBV target nucleic acid sequence. Amplification primers complementary tothis opposite strand of the HBV target nucleic acid sequence preferablyhave lengths of up to 100 bases, or more preferably 20-31 bases, orstill more preferably 20-21 bases. These primers are particularlypreferred for use as non-promoter primers (i.e., primers that arecomplementary to the opposite strand of the HBV template when comparedwith the promoter-primers). As disclosed herein, these primers have atleast 20 contiguous bases, allowing for substitution of nitrogenous baseanalogs, from a sequence substantially corresponding toGGAATTAGAGGACAAACGGGCAACATACCTTGATAATCCAGAAGAACCAATAAG (SEQ ID NO:4).Examples of particular amplification primers fulfilling these criteriainclude oligonucleotides having the sequences given by SEQ ID NOs:5-15.Even more preferred are primers having at least 20 contiguous bases,allowing for substitution of nitrogenous base analogs, from a sequencesubstantially corresponding toAGGACAAACGGGCAACATACCTTGATAATCCAGAAGAACCAATAAG (SEQ ID NO:142). Examplesof particular amplification primers fulfilling these criteria includeoligonucleotides having the sequences given by SEQ ID NOs:5-9, 11 and15.

It should be understood that the above-specified variable lengths of theamplification primers accommodate the presence of extraneous sequencesthat do not participate in target binding, and that may notsubstantially affect amplification or detection procedures. For example,promoter-primers useful for performing amplification reactions inaccordance with the invention have at least a minimal sequence thathybridizes to the HBV target nucleic acid, and a promoter sequencepositioned upstream of that minimal sequence. However, insertion ofsequences between the target binding sequence and the promoter sequencecould change the length of the primer without compromising its utilityin the amplification reaction. Additionally, the lengths of theamplification primers and detection probes are matters of choice as longas the sequences of these oligonucleotides conform to the minimalessential requirements for hybridizing the desired complementarysequence.

Tables 1 and 2 present specific examples of oligonucleotide sequencesthat were used as primers for amplifying HBV nucleic acids. Table 1presents the sequences of primers that were complementary to HBVsequences on one strand of nucleic acid. Table 2 presents the sequencesof both the HBV target-complementary sequences and the full sequencesfor promoter-primers that were used during development of the invention.Compared with the oligonucleotide sequences in Table 1, theoligonucleotide sequences in Table 2 are complementary to the oppositenucleic acid strand of the HBV genome.

TABLE 1 Polynucleotide Sequences of Amplification Primers Sequence (5′to 3′) Identifier TTGATAATCCAGAAGAACCAA SEQ ID NO: 5CTTGATAATCCAGAAGAACCA SEQ ID NO: 6 TACCTTGATAATCCAGAAGAACCA SEQ ID NO: 7GATAATCCAGAAGAACCAATAA SEQ ID NO: 8 ATAATCCAGAAGAACCAATAAG SEQ ID NO: 9AGAGGACAAACGGGCAACAT SEQ ID NO: 10 AGGACAAACGGGCAACATAC SEQ ID NO: 11GGAATTAGAGGACAAACGGGCAACATACCTT SEQ ID NO: 12TTAGAGGACAAACGGGCAACATACCTT SEQ ID NO: 13 GAGGACAAACGGGCAACATACCTTSEQ ID NO: 14 GACAAACGGGCAACATACCTT SEQ ID NO: 15

Table 2 presents HBV target-complementary oligonucleotide sequences (SEQID NOs:16-32) and the corresponding promoter-primer sequences (SEQ IDNOs:33-49). As indicated above, all promoter-primers included sequencescomplementary to an HBV target sequence at their 3′ ends, and a T7promoter sequence at their 5′ ends.

TABLE 2 Polynucleotide Sequences of Amplification Primers Sequence (5′to 3′) Identifier GTGTCTTGGCCAAAATTCGCAGTC SEQ ID NO: 16(HBV complementary primer) GTCCCCAACCTCCAATCACTCACCAA SEQ ID NO: 17(HBV complementary primer) CCCAACCTCCAATCACTCACCAAC SEQ ID NO: 18(HBV complementary primer) CTGTCCTCCAATTTGTCCTGGTTATC SEQ ID NO: 19(HBV complementary primer) CCAACCTCCTGTCCTCCAATTTGTCCT SEQ ID NO: 20(HBV complementary primer) CAACCTCCTGTCCTCCAATTTGTCCTG SEQ ID NO: 21(HBV complementary primer) CTGGATGTGTCTGCGGCGTT SEQ ID NO: 22(HBV complementary primer) GATGTGTCTGCGGCGTTTTATC SEQ ID NO: 23(HBV complementary primer) ATCATATTCCTCTTCATCCTGCT SEQ ID NO: 24(HBV complementary primer) ATATTCCTCTTCATCCTGCTGCT SEQ ID NO: 25(HBV complementary primer) ATATTCCTCTICATCCTGCTGCT SEQ ID NO: 26(HBV complementary primer) ATATTCCTCTTCATCCTGCTGCTA SEQ ID NO: 27(HBV complementary primer) ATTCCTCTTCATCCTGCTGCTAT SEQ ID NO: 28(HBV complementary primer) AATTTGTCCTGGTTATCGCTG SEQ ID NO: 29(HBV complementary primer) CCTGGTTATCGCTGGATG SEQ ID NO: 30(HBV complementary primer) CTGGTTATCGCTGGATGT SEQ ID NO: 31(HBV complementary primer) TGGTTATCGCTGGATGTG SEQ ID NO: 32(HBV complementary primer) AATTTAATACGACTCACTATAGGGAGA SEQ ID NO: 33GTGTCTTGGCCAAAATTCGCAGTC (T7 promoter-primer)AATTTAATACGACTCACTATAGGGAGA SEQ ID NO: 34 GTCCCCAACCTCCAATCACTCACCAA(T7 promoter-primer) AATTTAATACGACTCACTATAGGGAGA SEQ ID NO: 35CCCAACCTCCAATCACTCACCAAC (T7 promoter-primer)AATTTAATACGACTCACTATAGGGAGACTGTCCTCCAATT SEQ ID NO: 36 TGTCCTGGTTATC(T7 promoter-primer) AATTTAATACGACTCACTATAGGGAGA SEQ ID NO: 37CCAACCTCCTGTCCTCCAATTTGTCCT (T7 promoter-primer)AATTTAATACGACTCACTATAGGGAGA SEQ ID NO: 38 CAACCTCCTGTCCTCCAATTTGTCCTG(T7 promoter-primer) AATTTAATACGACTCACTATAGGGAGA SEQ ID NO: 39CTGGATGTGTCTGCGGCGTT (T7 promoter-primer) AATTTAATACGACTCACTATAGGGAGASEQ ID NO: 40 GATGTGTCTGCGGCGTTTTATC (T7 promoter-primer)AATTTAATACGACTCACTATAGGGAGA SEQ ID NO: 41 ATCATATTCCTCTTCATCCTGCT(T7 promoter-primer) AATTTAATACGACTCACTATAGGGAGA SEQ ID NO: 42ATATTCCTCTTCATCCTGCTGCT (T7 promoter-primer) AATTTAATACGACTCACTATAGGGAGASEQ ID NO: 43 ATATTCCTCTICATCCTGCTGCT (T7 promoter-primer)AATTTAATACGACTCACTATAGGGAGA SEQ ID NO: 44 ATATTCCTCTTCATCCTGCTGCTA(T7 promoter-primer) AATTTAATACGACTCACTATAGGGAGA SEQ ID NO: 45ATTCCTCTTCATCCTGCTGCTAT (T7 promoter-primer) AATTTAATACGACTCACTATAGGGAGASEQ ID NO: 46 AATTTGTCCTGGTTATCGCTG (T7 promoter-primer)AATTTAATACGACTCACTATAGGGAGA SEQ ID NO: 47 CCTGGTTATCGCTGGATG(T7 promoter-primer) AATTTAATACGACTCACTATAGGGAGA SEQ ID NO: 48CTGGTTATCGCTGGATGT (T7 promoter-primer) AATTTAATACGACTCACTATAGGGAGASEQ ID NO: 49 TGGTTATCGCTGGATGTG (T7 promoter-primer)

Preferred sets of primers for amplifying HBV sequences in a TMA reactioninclude a first primer that hybridizes an HBV target sequence (such asone of the primers listed in Table 2) and a second primer that iscomplementary to the sequence of an extension product of the firstprimer (such as one of the primer sequences listed in Table 1). In ahighly preferred embodiment, the first primer is a promoter-primer thatincludes a T7 promoter sequence at its 5′ end.

Preferred Detection Probes

Another aspect of the invention relates to oligonucleotides that can beused as hybridization probes for detecting HBV nucleic acids. Methodsfor amplifying a target nucleic acid sequence present in the nucleicacid of HBV can include an optional further step for detectingamplicons. This procedure for detecting HBV nucleic acids includes astep for contacting a test sample with a hybridization assay probe thatpreferentially hybridizes to the target nucleic acid sequence, or thecomplement thereof, under stringent hybridization conditions, therebyforming a probe:target duplex that is stable for detection. Next thereis a step for determining whether the hybrid is present in the testsample as an indication of the presence or absence of HBV nucleic acidsin the test sample. This may involve detecting the probe:target duplex.

Hybridization assay probes useful for detecting HEY nucleic acidsequences include a sequence of bases substantially complementary to anHBV target nucleic acid sequence. Thus, probes of the inventionhybridize one strand of an HBV target nucleic acid sequence, or thecomplement thereof. These probes may optionally have additional basesoutside of the targeted nucleic acid region which may or may not becomplementary to HBV nucleic acid.

Preferred probes are sufficiently homologous to the target nucleic acidto hybridize under stringent hybridization conditions corresponding toabout 60° C. when the salt concentration is in the range of 0.6-0.9 M.Preferred salts include lithium chloride, but other salts such as sodiumchloride and sodium citrate also can be used in the hybridizationsolution. Example high stringency hybridization conditions arealternatively provided by 0.48 M sodium phosphate buffer, 0.1% sodiumdodecyl sulfate, and 1 mM each of EDTA and EGTA, or by 0.6 M LiCl, 1%lithium lauryl sulfate, 60 mM lithium succinate and 10 mM each of EDTAand EGTA.

Certain probes that are preferred for detecting HBV nucleic acidsequences have target-complementary sequences in the length range offrom 17-23 nucleotides. Highly preferred probes that may be used fordetecting HBV nucleic acids have target-complementary sequences 23nucleotides, more preferably 22, more preferably 20, more preferably 19,more preferably 18, or more preferably 17 nucleotides in length. Ofcourse, these target-complementary sequences may be linear sequences, ormay be contained in the structure of a molecular beacon or otherconstruct having one or more optional nucleic acid sequences that arenon-complementary to the HBV target sequence that is to be detected. Asindicated above, probes may be made of DNA, RNA, a combination DNA andRNA, a nucleic acid analog, or contain one or more modified nucleosides(e.g., a ribonucleoside having a 2′-O-methyl substitution to theribofuranosyl moiety).

Examples of probes that can be used to carry out the assay disclosedherein include at least 17, more preferably 17-23 or still morepreferably 17-19 contiguous nucleotides contained within the sequencegiven by ACGGGCAACATACCTTGATAGTCCAGAAGAACCAACAAGAAGATGAGGCATAGCAGCAGGATGCAGAGGAA (SEQ ID NO:67), or the complement thereof, allowing forthe presence of RNA and DNA equivalents and the substitution ofnucleotide analogs. Certain preferred probes in accordance with thepresent invention include a detectable label. In one embodiment thislabel is an acridinium ester that is joined to the probe by means of anon-nucleotide linker. For example, detection probes can be labeled withchemiluminescent acridinium ester compounds that are attached via alinker substantially as described in U.S. Pat. No. 5,585,481; and inU.S. Pat. No. 5,639,604, particularly as described at column 10, line 6to column 11, line 3, and in Example 8. The disclosures contained inthese patent documents are hereby incorporated by reference. When thehybridization probe is labeled with an acridinium ester moiety via aninternally disposed linker, the probe is preferably stored in thepresence of a “protection probe” in accordance with U.S. Pat. No.6,245,519, the disclosure of which is hereby incorporated by reference.For example, if the probe is 19 bases long and has an acridinium esterjoined between positions 6 and 7, the corresponding protection probe canbe 16 bases long to provide the necessary differential Tm. In thisinstance we used a protection probe that had 3 complementary bases onone side, and 13 complementary bases on the other side of an acridiniumester label that was linked to the probe. Of course, other sequencevariations may be envisioned.

Table 3 presents the sequences of hybridization probes that were usedfor detecting HBV amplicons. Since alternative probes for detecting HBVnucleic acid sequences can hybridize to the opposite-sense strand ofHBV, the present invention also includes oligonucleotides that arecomplementary to the sequences presented in the table.

TABLE 3 Polynucleotide Sequences of HBV Detection Probes Sequence (5′to 3′) Identifier AGCAGGAUGAAGAGGAA SEQ ID NO: 50 GCAGCAGGAUGAAGAGGASEQ ID NO: 51 GCAGCAGGATGAAGAGG SEQ ID NO: 52 UGAGGCAUAGCAGCAGGASEQ ID NO: 53 GAAGATGAGGCATAGCAGC SEQ ID NO: 54 ACAAGAAGAUGAGGCAUAGCAGCSEQ ID NO: 55 AGAAGAUGAGGCAUAGCAG SEQ ID NO: 56 AAGAAGAUGAGGCAUAGCSEQ ID NO: 57 ACAAGAAGATGAGGCATA SEQ ID NO: 58 CCAACAAGAAGAUGAGGCSEQ ID NO: 59 AIUCCAGAAGAACCAACAAGAAG SEQ ID NO: 60AIUCCAGAAGAACCAACAAGAAG SEQ ID NO: 61 CCAGAAGAACCAACAAGAAG SEQ ID NO: 62CCUUGAUAGUCCAGAAGAACCA SEQ ID NO: 63 CCUUGAUAGUCCAGAAGAACCAASEQ ID NO: 64 ACGGGCAACAUACCUUG SEQ ID NO: 65 CGGGCAACAUACCUUGSEQ ID NO: 66

In some embodiments of the invention, the probe sequence used fordetecting HBV amplicons includes a methoxy backbone, or at least onemethoxy linkage in the nucleic acid backbone.

Selection and Use of Capture Oligonucleotides

Preferred capture oligonucleotides include a first sequence that iscomplementary to an HBV sequence (i.e., an “HBV target sequence”)covalently attached to a second sequence (i.e., a “tail” sequence) thatserves as a target for immobilization on a solid support. Any backboneto link the base sequence of a capture oligonucleotide may be used. Incertain preferred embodiments the capture oligonucleotide includes atleast one methoxy linkage in the backbone. The tail sequence, which ispreferably at the 3′ end of a capture oligonucleotide, is used tohybridize to a complementary base sequence to provide a means forcapturing the hybridized target HBV nucleic acid in preference to othercomponents in the biological sample.

Although any base sequence that hybridizes to a complementary basesequence may be used in the tail sequence, it is preferred that thehybridizing sequence span a length of about 5-50 nucleotide residues.Particularly preferred tail sequences are substantially homopolymeric,containing about 10 to about 40 nucleotide residues, or more preferablyabout 14 to about 30 residues. A capture oligonucleotide according tothe present invention may include a first sequence that specificallybinds an HBV target polynucleotide, and a second sequence thatspecifically binds an oligo(dT) stretch immobilized to a solid support.

Using the components illustrated in FIG. 1, one assay for detecting HBVsequences in a biological sample includes the steps of capturing thetarget nucleic acid using the capture oligonucleotide, amplifying thecaptured target region using at least two primers, and detecting theamplified nucleic acid by first hybridizing the labeled probe to asequence contained in the amplified nucleic acid and then detecting asignal resulting from the bound labeled probe.

The capturing step preferably uses a capture oligonucleotide where,under hybridizing conditions, one portion of the capture oligonucleotidespecifically hybridizes to a sequence in the target nucleic acid and atail portion serves as one component of a binding pair, such as a ligand(e.g., a biotin-avidin binding pair) that allows the target region to beseparated from other components of the sample. Preferably, the tailportion of the capture oligonucleotide is a sequence that hybridizes toa complementary sequence immobilized to a solid support particle.Preferably, first, the capture oligonucleotide and the target nucleicacid are in solution to take advantage of solution phase hybridizationkinetics. Hybridization produces a capture oligonucleotide:targetnucleic acid complex which can bind an immobilized probe throughhybridization of the tail portion of the capture oligonucleotide with acomplementary immobilized sequence. Thus, a complex comprising a targetnucleic acid, capture oligonucleotide and immobilized probe is formedunder hybridization conditions. Preferably, the immobilized probe is arepetitious sequence, and more preferably a homopolymeric sequence(e.g., poly-A, poly-T, poly-C or poly-G), which is complementary to thetail sequence and attached to a solid support. For example, if the tailportion of the capture oligonucleotide contains a poly-A sequence, thenthe immobilized probe would contain a poly-T sequence, although anycombination of complementary sequences may be used. The captureoligonucleotide may also contain “spacer” residues, which are one ormore bases located between the base sequence that hybridizes to thetarget and the base sequence of the tail that hybridizes to theimmobilized probe. Any solid support may be used for binding the targetnucleic acid:capture oligonucleotide complex. Useful supports may beeither matrices or particles free in solution (e.g., nitrocellulose,nylon, glass, polyacrylate, mixed polymers, polystyrene, silanepolypropylene and, preferably, magnetically attractable particles).Methods of attaching an immobilized probe to the solid support are wellknown. The support is preferably a particle which can be retrieved fromsolution using standard methods (e.g., centrifugation, magneticattraction of magnetic particles, and the like). Preferred supports areparamagnetic monodisperse particles (i.e., uniform in size ±about 5%).

Retrieving the target nucleic acid:capture oligonucleotide:immobilizedprobe complex effectively concentrates the target nucleic acid (relativeto its concentration in the biological sample) and purifies the targetnucleic acid from amplification inhibitors which may be present in thebiological sample. The captured target nucleic acid may be washed one ormore times, further purifying the target, for example, by resuspendingthe particles with the attached target nucleic acid:captureoligonucleotide:immobilized probe complex in a washing solution and thenretrieving the particles with the attached complex from the washingsolution as described above. In a preferred embodiment, the capturingstep takes place by sequentially hybridizing the capture oligonucleotidewith the target nucleic acid and then adjusting the hybridizationconditions to allow hybridization of the tail portion of the captureoligonucleotide with an immobilized complementary sequence (e.g., asdescribed in PCT No. WO 98/50583). After the capturing step and anyoptional washing steps have been completed, the target nucleic acid canthen be amplified. To limit the number of handling steps, the targetnucleic acid optionally can be amplified without releasing it from thecapture oligonucleotide.

Two different regions of the HBV nucleic acid sequence are describedherein as targets for capture oligonucleotides, with each region beinglocated outside one of the oppositely disposed boundaries that definethe HBV nucleic acid sequence that may be amplified by the methodsdescribed herein. The sequences of capture oligonucleotides thathybridize one of these regions are contained within a sequencesubstantially corresponding toAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGATCACCCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAAC (SEQ ID NO:68). Preferred lengthsfor the HBV-complementary portions of the capture oligonucleotides fallin the range of from 20-40 nucleotides, more preferably 25-30nucleotides. Examples of useful capture oligonucleotides falling in thiscategory include oligonucleotides having sequences given by SEQ IDNOs:70-76. The sequences of capture oligonucleotides that hybridize theother region of the HBV nucleic acid sequence are contained within asequence substantially corresponding toCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTT (SEQ ID NO:69). Preferred sizes for these captureoligonucleotides fall in the range of from 25-50 nucleotides, morepreferably 25-32 nucleotides in length. Examples of useful captureoligonucleotides falling in this category include oligonucleotideshaving the HBV-complementary sequences of SEQ ID NOs:77-87. Usefulcapture oligonucleotides may contain mismatches to the above-indicatedsequences, as long as the mismatched sequences hybridize to the HBVnucleic acid containing the sequence that is to be amplified. Eachcapture oligonucleotide described herein included one of theHBV-complementary sequences presented in Table 4 linked to a poly-(dA)tail at its 3′ end. With the exception of the oligonucleotidesidentified by SEQ ID NO:71 and SEQ ID NO:80, all of the captureoligonucleotides also included three thymidine nucleotides interposedbetween the HBV-complementary sequence and the poly-(dA) tail. Asindicated below, the presence of these thymidine nucleotides is notbelieved to be essential for success of the capture procedure. Notably,the presence of a “C” nucleobase, rather than the naturally occurring“G” nucleobase, at position 16 of capture oligonucleotide of SEQ IDNO:86 demonstrated that at least one base substitution is permitted inthe capture oligonucleotides of the present invention.

TABLE 4 Polynucleotide Sequences of HBV-ComplementaryPortions of Capture Oligonucleotides Sequence (5′ to 3′) IdentifierAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGG SEQ ID NO: 70GTGGTGGACTTCTCTCAATTTTCTAGGGGG SEQ ID NO: 71 GGATCACCCGTGTGTCTTGGSEQ ID NO: 72 GTGTCTTGGCCAAAATTCGCAGTCC SEQ ID NO: 73GCCAAAATTCGCAGTCCCCAACCTCCAATCACTCAC SEQ ID NO: 74 CAACGCCAAAATTCGCAGTCCCCAACCTCCA SEQ ID NO: 75 TTCGCAGTCCCCAACCTCCAATCACTCSEQ ID NO: 76 CGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTT SEQ ID NO: 77 CAGTCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTT SEQ ID NO: 78 CAGTGGTCGTGGCTCAGTTTACTAGTGCCATTTGTTCAGTG SEQ ID NO: 79TGGCTCAGTTTACTAGTGCCATTTGTTCAGTG SEQ ID NO: 80TGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGT SEQ ID NO: 81GGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCG SEQ ID NO: 82GGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCG SEQ ID NO: 83 TAGGGCGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCG SEQ ID NO: 84 TAGGGCTTTCCCCCGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCC SEQ ID NO: 85GGGCTTTCCCCCACTCTTTGGCTTT SEQ ID NO: 86 GGGCTTTCCCCCACTGTTTGGCTTTSEQ ID NO: 87Preferred Methods for Amplifying and Detecting HBV PolynucleotideSequences

Preferred methods of the present invention are described and illustratedby the Examples presented below. FIG. 1 schematically illustrates onesystem that may be used for detecting a target region of the HBV genome(shown by a thick solid horizontal line). This system includes fouroligonucleotides (shown by the shorter solid lines): one captureoligonucleotide that includes a sequence that hybridizes specifically toan HBV sequence in the target region and a tail (“T”) that hybridizes tocomplementary sequence immobilized on a solid support to capture thetarget region present in a biological sample; one T7 promoter-primerwhich includes a sequence that hybridizes specifically to an HBVsequence in the target region and a T7 promoter sequence (“P”) which,when double-stranded, serves as a functional promoter for T7 RNApolymerase; one non-T7 primer which includes a sequence that hybridizesspecifically to a first strand cDNA made from the target region sequenceusing the T7 promoter-primer; and one labeled probe which includes asequence that hybridizes specifically to a portion of the target regionthat is amplified using the two primers.

As indicated above, amplifying the captured target region using the twoprimers can be accomplished by any of a variety of known nucleic acidamplification reactions that will be familiar to those having anordinary level of skill in the art. In a preferred embodiment, atranscription-associated amplification reaction, such as TMA, isemployed. In such an embodiment, many strands of nucleic acid areproduced from a single copy of target nucleic acid, thus permittingdetection of the target by detecting probes that are bound to theamplified sequences. Preferably, transcription-associated amplificationuses two types of primers (one being referred to as a promoter-primerbecause it contains a promoter sequence, labeled “P” in FIG. 1, for anRNA polymerase) two enzymes (a reverse transcriptase and an RNApolymerase), and substrates (deoxyribonucleoside triphosphates,ribonucleoside triphosphates) with appropriate salts and buffers insolution to produce multiple RNA transcripts from a nucleic acidtemplate.

Referring to FIG. 1, during transcription-mediated amplification, thecaptured target nucleic acid is hybridized to a first primer shown as aT7 promoter-primer. Using reverse transcriptase, a complementary DNAstrand is synthesized from the T7 promoter-primer using the target DNAas a template. A second primer, shown as a non-T7 primer, hybridizes tothe newly synthesized DNA strand and is extended by the action of areverse transcriptase to form a DNA duplex, thereby forming adouble-stranded T7 promoter region. T7 RNA polymerase then generatesmultiple RNA transcripts by using this functional T7 promoter. Theautocatalytic mechanism of TMA employs repetitive hybridization andpolymerization steps following a cDNA synthesis step using the RNAtranscripts as templates to produce additional transcripts, therebyamplifying target region-specific nucleic acid sequences.

The detecting step uses at least one detection probe that bindsspecifically to the amplified RNA transcripts or amplicons describedabove. Preferably, the detection probe is labeled with a label that canbe detected using a homogeneous detection system. For example, thelabeled probe can be labeled with an acridinium ester compound fromwhich a chemiluminescent signal may be produced and detected, asdescribed above. Alternatively, the labeled probe may comprisefluorophore and quencher moieties. A molecular beacon is one embodimentof such a labeled probe that may be used in a homogeneous detectionsystem.

Multiplex Amplification Reactions

A convenient testing format for detecting multiple analytepolynucleotides involves conducting simultaneous amplification reactionsusing different primer sets, wherein amplicons synthesized in thereaction are detected by hybridization. In this regard, Gen-ProbeIncorporated (San Diego, Calif.) has developed an HIV-1/HCV test thatdetects HIV-1 and/or HCV (Hepatitis C Virus) nucleic acids in asingle-tube multiplex format using three key steps. In an initial samplepreparation procedure plasma is treated with detergent to release viralgenomic RNA, target-specific oligonucleotides are hybridized to thetarget and captured onto magnetic microparticles which are separatedfrom plasma in a magnetic field. Next, a transcription-basedpolynucleotide amplification system is employed to amplify HIV-1 and/orHCV target RNA in a single reaction. Finally, detection is accomplishedusing nucleic acid hybridization of chemiluminescent probes that arecomplementary to the HIV-1 or HCV amplicons. If the assay gives apositive result, discriminatory tests are performed to differentiatebetween the two viruses.

Oligonucleotides that were particularly used for amplifying anddetecting HIV-1 nucleic acids in the procedures described below had thefollowing sequences. Amplification primers specific for one strand ofthe HIV-1 nucleic acid included the sequences of CGGGCGCCACTGCTAGAGATTTT(SEQ ID NO:98) and GTTTGTATGTCTGTTGCTATTATGTCTA (SEQ ID NO:99), witheach primer further including an optional upstream promoter sequence togive the corresponding promoter-primers ofAATTTAATACGACTCACTATAGGGAGACGGGCGCCACTGCTAGAGATTTT (SEQ ID NO:100) andAATTTAATACGACTCACTATAGGGAGAGTTTGTATGTCTGTTGCTATTATGTCTA (SEQ ID NO:101).Opposite strand primers that were used during the development of theinvention had the sequences of GCCTCAATAAAGCTTGCC (SEQ ID NO:102) andACAGCAGTACAAATGGCAG (SEQ ID NO:103). Although not used for obtaining themultiplex data presented herein, preferred alternative opposite strandprimers include GACAGCAGTACAAATGGCAG (SEQ ID NO:104) andAGACAGCAGTACAAATGGCAG (SEQ ID NO:105). Probes for detecting HIVamplicons had the sequences given by CCACAATTTTAAAAGAAAAGGG (SEQ IDNO:106) (labeled with AE between nucleotide positions 7 and 8),CUGGUAICUAGAGAUCCCUC (SEQ ID NO:107) (labeled with AE between nucleotidepositions 9 and 10), and GGATTGGIIIGTACAGTGC (SEQ ID NO:108) (labeledwith AE between nucleotide positions 5 and 6). A probe having thesequence of CCACAAGCUUAGAAGAUAGAGAGG (SEQ ID NO:109) (labeled with AEbetween nucleotide positions 10 and 11) was used for detecting aninternal control amplicon. All of these probes were prepared using2′-0Me analogs, except for the final position which was occupied by aDNA nucleotide, thereby confirming that the probes can be flexiblyprepared using a combination of RNA and DNA equivalent bases, baseanalogs and nucleotide backbone analogs. Probe protectionoligonucleotides had the sequences of GGATCTCTAGCTACC (SEQ ID NO:110),CCCTTTTCTTTTAAAATTGTGG (SEQ ID NO:111), and CTATCTTCTAAGCTTG (SEQ IDNO:112). Capture oligonucleotides included the HIV-complementarysequences of ACUGACGCUCUCGCACCCAUC (SEQ ID NO:113) andUCUGCUGUCCCUGUAAUAAACCCG (SEQ ID NO:114), and were prepared using 2′-OMeanalogs and joined at their 3′-ends to a tail sequence of A₃₀ with threeT residues interposed therebetween.

Preferred oligonucleotides used for amplifying and detecting HCV nucleicacids had the following sequences. An amplification primer specific forone strand of the HCV nucleic acid included the sequence ofAGTACCACAAGGCCTTTCGCIACCCAAC (SEQ ID NO:115), and further included anoptional upstream promoter sequence to give the correspondingpromoter-primer ofAATTTAATACGACTCACTATAGGGAGAAGTACCACAAGGCCTTTCGCIACCCAAC (SEQ ID NO:116).Opposite strand primers that were used during the development of thepresent invention had the sequences of CTAGCCATGGCGTTAGTA (SEQ IDNO:117) and CTACTGTCTTCACGCAGAAAGCG (SEQ ID NO:118). Probes fordetecting HCV amplicons had the sequences given byCCCGGGAGAGCCAUAGUGGUCT (SEQ ID NO:119) (labeled with AE betweennucleotide positions 14 and 15), and CTGCGGAACCGGTGAGTAC (SEQ ID NO:120)(labeled with AE between nucleotide positions 13 and 14). Detection ofHCV amplicons was facilitated by the use of a helper probe having thesequence of AGCCUCCAGGACCCCCCCT (SEQ ID NO:121). As above, these probeswere prepared using 2′-OMe analogs, except for the final position whichwas occupied by a DNA nucleotide, thereby confirming that the probes canbe flexibly prepared using a combination of RNA and DNA equivalentbases, base analogs and nucleotide backbone analogs. Probe protectionoligonucleotides had the sequences of GACCACTATGGCTC (SEQ ID NO:122) andGTACTCACCGGTTC (SEQ ID NO:123). Capture oligonucleotides included theHCV-complementary sequences of GGGCACUCGCAAGCACCCU (SEQ ID NO:124) andCAUGGUGCACGGUCUACG (SEQ ID NO:125), and were prepared using 2′-OMeanalogs and joined at their 3′-ends to a tail sequence of A₃₀ with threeT residues interposed therebetween.

Notably, the above-described oligonucleotides can be used under theconditions described herein for single-analyte detection. Indeed, theoligonucleotides that were used for amplifying and detecting HIV-1 canbe used in a stand-alone assay for detecting HIV-1, and theoligonucleotides that were used for amplifying and detecting HCV can beused in a stand-alone assay for detecting HCV. Each of these alternativestand-alone assays represents a preferred embodiment of the presentinvention. Moreover, the oligonucleotides that were used for amplifyingand detecting HIV-1 have been used in combination with theoligonucleotides that were used for amplifying and detecting HCV in amultiplex reaction. Again, this combination represents another preferredembodiment of the present invention. Finally, as disclosed in numerousexamples herein, the HIV-1 and HCV amplifying and detectingoligonucleotides have been used in combination to prepare an assaycapable of amplifying and detecting any or all of the HIV-1, HCV and HBVanalytes.

As the number of different primer sets in a multiplex amplificationreaction increases, with each set of primers being specific for adifferent analyte polynucleotide, there is an increased opportunity forundesired interaction among primers, and between primers and irrelevantamplicons. The most highly preferred primer sequences disclosed hereincan be used in reactions that specifically amplify only HBV nucleicacids, and also can be used as reagents in a single polynucleotideamplification reaction that is additionally capable of amplifyingvirus-specific sequences from HIV-1 and HCV.

Kits for Detecting HBV Nucleic Acids

The present invention also embraces kits for performing polynucleotideamplification reactions using viral nucleic acid templates. Certainpreferred kits contain a pair of oligonucleotide primers that may beused for amplifying HBV nucleic acids in an in vitro amplificationreaction. Exemplary kits include first and second amplificationoligonucleotides that are complementary to opposite strands of the HBVnucleic acid sequence that is to be amplified. The first amplificationoligonucleotide, which is also referred to as the “first strand” primer,includes a downstream sequence complementary to one strand of the HBVnucleic acid, and optionally an upstream sequence that is notcomplementary to that HBV nucleic acid. Preferably, the firstamplification oligonucleotide has a length of up to 100 bases, morepreferably a length of from 20-60 bases, and has an HBV-complementarysequences that includes at least 20 contiguous bases, more preferably20-50 contiguous bases, still more preferably 20-24 contiguous bases,allowing for substitution of one or more nucleotides or nitrogenous baseanalogs, contained within a sequence substantially corresponding to SEQID NO:2. Primers falling within this group include SEQ ID NOs:22-28, andthe corresponding T7 promoter-primers of SEQ ID NOs:39-45. The secondamplification oligonucleotide preferably has a length of up to 100bases, or more preferably 20-31 bases, or still more preferably 20-21bases. This second primer, which is also referred to as the “secondstrand” primer, has at least 20 contiguous bases, more preferably 20-54contiguous bases, more preferably 20-21 contiguous bases, allowing forsubstitution of nucleotides or nitrogenous base analogs, containedwithin a sequence substantially corresponding to SEQ ID NO:4.Amplification primers fulfilling these criteria include oligonucleotideshaving the sequences given by SEQ ID NOs:5-15. The kits may furthercontain one or more oligonucleotide detection probes. These probes mayinclude at least 17 contiguous nucleotides, more preferably 17-23contiguous nucleotides or still more preferably 17-19 contiguousnucleotides that are contained within the sequence given by SEQ IDNO:67, or the complement thereof, allowing for the presence of RNA andDNA equivalents and the substitution of nucleotide analogs. Particularlypreferred probes include those having the sequences of SEQ ID NO:50, SEQID NO:57 and SEQ ID NO:54, although other probes disclosed herein can beused, and are intended to fall within the scope of the invention. Ofcourse, the complements of these sequences also can be used as probesfor detecting HBV sequences. Other probes that may be included in thekit are molecular beacon hybridization probes of the type describedunder Example 10. Still other kits in accordance with the invention mayadditionally include capture oligonucleotides for purifying HBV templatenucleic acids away from other species prior to amplification. Examplecapture oligonucleotides have lengths of up to 100 nucleotides, andHBV-complementary portions that fall in the range of from at least 20contiguous nucleotides, more preferably 20-40 contiguous nucleotides, orstill more preferably 25-30 contiguous nucleotides of SEQ ID NO:68.Examples of useful capture oligonucleotides falling in this categoryinclude oligonucleotides having sequences given by SEQ ID NOs:70-76.Alternative capture oligonucleotides have lengths of up to 100nucleotides, and HBV-complementary portions that fall in the range offrom at least 25 contiguous nucleotides, more preferably 25-50contiguous nucleotides, or still more preferably 25-32 contiguousnucleotides of SEQ ID NO:69. Examples of useful capture oligonucleotidesfalling in this category include oligonucleotides having sequences givenby SEQ ID NOs:77-87. Indeed, kits useful for practicing the inventedmethod of detecting HBV nucleic acids may include, in packagedcombination with one another, essentially any of the amplificationoligonucleotide compositions and/or detection probe compositions and/orcapture oligonucleotide compositions disclosed herein.

Other preferred kits in accordance with the invention include primersfor amplifying nucleic acids of viral targets other than HBV, but thatcan be used in combination with the primers for amplifying HBV targetnucleic acids. For example, the primers for amplifying HBV targetnucleic acids may be in packaged combination with primers that can beused for amplifying HIV-1 and/or HCV target nucleic acids. Exemplaryprimers for amplifying HIV-1 and HCV are disclosed herein. In aparticularly preferred embodiment, the kit includes primers foramplifying HBV, HIV-1 and HCV. Still other kits include primers that canbe used for amplifying only one of either HIV-1 or HCV target nucleicacids. Yet other kits include the primers for amplifying both HIV-1 andHCV, but do not include primers for amplifying HBV nucleic acids. Thus,it should be clear that many different kit configurations are embracedby the present invention. Probes, primers and capture oligonucleotidesequences for amplifying and detecting HIV-1 and HCV nucleic acids aredisclosed herein.

The general principles of the present invention may be more fullyappreciated by reference to the following non-limiting Examples.

Example 1 describes procedures that identified hybridization probeswhich subsequently were used in assays for detecting HBV nucleic acids.One of seven related synthetic oligonucleotides served as a target forbinding of candidate probes in this procedure.

EXAMPLE 1 Oligonucleotide Probes for Detecting HBV

Synthetic HBV target oligonucleotides were prepared according tostandard laboratory procedures using RNA or 2′-0Me backbones. Thesetargets had the following sequences: UUCCUCUUCAUCCUGCU (SEQ ID NO:88);UUCCUCUUCAUCCUGCUGCUAUGCCUCAUCUUCUU (SEQ ID NO:89);UUCCUCUGCAUCCUGCUGCUAUGCCUCAUCUUCUUGUUG (SEQ ID NO:90);GCCUCAUCUUCUUGUUGG (SEQ ID NO:91); CUCAUCUUCUUGUUGGUUCUUCUGGACUAUCAAGG(SEQ ID NO:92); UUGGUUCUUCUGGACUAUCAAGG (SEQ ID NO:93); andCAAGGUAUGUUGCCCGU (SEQ ID NO:94). Candidate probes for hybridizing thesynthetic HBV targets were prepared using 2′-OMe nucleotides and had thesequences given in Table 3. Notably, position 8 of the target sequenceidentified by SEQ ID NO:90 was occupied by a G moiety which ischaracteristic of the HBV genotype B sequence.

Hybridization reactions consisted of 100 μl volumes of hybridizationbuffer containing amounts of AE-labeled probe corresponding to 5×10⁵RLUs and 10 μl containing 100 fmoles of the synthetic HBV targetoligonucleotide. Negative control reactions omitted the HBV targetoligonucleotide. The probes listed in Table 5 were each labeled with anAE moiety joined to the oligonucleotide structure by an internallydisposed non-nucleotide linker according to procedures described in U.S.Pat. Nos. 5,585,481 and 5,639,604, the disclosures of these patentshaving been incorporated by reference hereinabove. Use of the differentlinker positions confirmed the versatility of this labeling technique.More particularly, the linker on the probe of SEQ ID NO:50 was locatedbetween positions 8 and 9, the linker on the probe of SEQ ID NO:51 waslocated between positions 5 and 6, the linker on the probe of SEQ IDNO:52 was located between positions 5 and 6, the linker on the probe ofSEQ ID NO:53 was located between positions 11 and 12, the linker on theprobe of SEQ ID NO:54 was located between positions 6 and 7 or betweenpositions 13 and 14, the linker on the probe of SEQ ID NO:55 was locatedbetween positions 17 and 18 or between positions 8 and 9, the linker onthe probe of SEQ ID NO:56 was located between positions 14 and 15, thelinker on the probe of SEQ ID NO:57 was located between positions 8 and9, the linker on the probe of SEQ ID NO:58 was located between positions10 and 11, the linker on the probe of SEQ ID NO:59 was located betweenpositions 13 and 14, the linker on the probe of SEQ ID NO:60 was locatedbetween positions 8 and 9, the linker on the probe of SEQ ID NO:61 waslocated between positions 9 and 10, the linker on the probe of SEQ IDNO:62 was located between positions 6 and 7, the linker on the probe ofSEQ ID NO:63 was located between positions 14 and 15, the linker on theprobe of SEQ ID NO:64 was located between positions 17 and 18, thelinker on the probe of SEQ ID NO:65 was located between positions 5 and6, and the linker on the probe of SEQ ID NO:66 was located betweenpositions 8 and 9. Probe hybridizations were performed in 200 μl of asolution containing 0.05M lithium succinate (pH 5), 0.6M LiCl, 1% (w/v)lithium lauryl sulfate, 10 mM EDTA, 10 mM EGTA, at 60° C. for 15minutes. Hybridization reactions were followed by addition of 300 μl of0.15 M sodium tetraborate (pH 8.5), and 1% TRITON X-100 (Union CarbideCorporation; Danbury, Conn.). These mixtures were first incubated at 60°C. for 10 minutes to inactivate unhybridized probe, and cooled to roomtemperature thereafter. Chemiluminescence due to hybridized probe ineach sample was assayed using a LEADER I luminometer (Gen-ProbeIncorporated) configured for automatic injection of 1 mM nitric acid and0.1% (v/v) hydrogen peroxide, followed by injection of a solutioncontaining 1 N sodium hydroxide. Results for the chemiluminescentreactions were measured in relative light units (RLU). Sample resultsfrom this procedure are presented in Table 5.

TABLE 5 Probe Hybridization Results Synthetic Negative HybridizationProbe HBV Target Control (RLU) Signal (RLU) SEQ ID NO: 50 SEQ ID NO: 88825 480,563 SEQ ID NO: 51 SEQ ID NO: 90 1004  443,434 SEQ ID NO: 52 SEQID NO: 89 785 423,935 SEQ ID NO: 53 SEQ ID NO: 90 945 447,499 SEQ ID NO:54 SEQ ID NO: 90 623 501,532 SEQ ID NO: 55 SEQ ID NO: 90 427 458,790 SEQID NO: 56 SEQ ID NO: 90 447 392,360 SEQ ID NO: 57 SEQ ID NO: 90 394659,028 SEQ ID NO: 58 SEQ ID NO: 90 516 497,271 SEQ ID NO: 59 SEQ ID NO:91 698 476,324 SEQ ID NO: 60 SEQ ID NO: 92 407 242,711 SEQ ID NO: 61 SEQID NO: 92 472 220,587 SEQ ID NO: 62 SEQ ID NO: 92 447 206,988 SEQ ID NO:63 SEQ ID NO: 93 371 99,625 SEQ ID NO: 64 SEQ ID NO: 93 386 79,736 SEQID NO: 65 SEQ ID NO: 94 415 14,415 SEQ ID NO: 66 SEQ ID NO: 94 43241,528

The results presented in Table 5 showed that each probe gave a lowbackground signal and at least a moderate level of positive reactionwith the HBV target oligonucleotide. However, some of the probes gaveresults that were substantially better than others, thereby defining apreferred target region for probe binding in the HBV sequence. Moreparticularly, with the exception of the probes of SEQ ID NOs:65-66, allof the probes gave negative control values that were less than 0.5% ofthe corresponding probe hybridization signals. Based on this pattern,preferred probes for hybridizing and detecting HBV amplicons include17-23 contiguous nucleotides contained within the sequence given byCCTTGATAGTCCAGAAGAACCAACAAGAAGATGAGGCATAGCAGCAGGATGCAGAG GAA (SEQ IDNO:95), or the complement thereof, allowing for RNA and DNA equivalentsand the presence of nucleotide analogs. Notably, while the resultpresented for the probe of SEQ ID NO:54 was for the oligonucleotidehaving a linker located between positions 6 and 7, substantiallyidentical results were obtained for a probe having an identicalnucleobase sequence with a linker located between positions 13 and 14.Each of these alternatives represents a preferred embodiment of theinvented probe. Further, while the result presented in the table for theprobe of SEQ ID NO:55 was for the oligonucleotide having a linkerlocated between positions 17 and 18, good, but somewhat lower valueswere obtained for a probe having an identical nucleobase sequence with alinker located between positions 8 and 9. Each of these alternativesalso represents a preferred embodiment of the invented probe. Indeed,the positioning of any detectable label joined to any of theabove-described probes can be varied and still fall within the scope ofthe invention.

Hybridization assay probes having the sequences of SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54 (using a combination of separate oligonucleotideshaving the two above-described label positions), SEQ ID NO:57, and SEQID NO:58 were subsequently used for demonstrating that a range ofamplification primers and capture oligonucleotides could detect HBVnucleic acids in biological samples. Probes having these sequences ortheir complements, allowing for the presence of RNA and DNA equivalentsand nucleotide analog substitutions, each represent particularlypreferred embodiments of the invention.

Preferred primer combinations for amplifying HBV nucleic acids wereidentified in a series of procedures that employed HBV virions as thesource of nucleic acid templates. Promoter-primers and opposite strandprimers were screened in combination using the method described below.Although these procedures were particularly carried out using aTranscription Mediated Amplification (TMA) protocol, the primersdisclosed herein may be used to produce amplicons by alternative invitro nucleic acid amplification methods that will be familiar to thosehaving an ordinary level of skill in the art.

Example 2 describes the methods that identified useful amplificationprimers. In addition to the HBV-specific target captureoligonucleotides, amplification primers and probes described in thisprocedure, the reactions also included the above-describedoligonucleotides specific for the HIV-1 and HCV analytes. The HIV-1 andHCV oligonucleotides did not substantially contribute to detection ofthe HBV analyte.

EXAMPLE 2 Identification of Amplification Primers

HBV genotype A virions served as the source of HBV template sequences inamplification reactions that employed paired sets of primers. TMAreactions were carried out essentially as described by Kacian et al., inU.S. Pat. No. 5,399,491, the disclosure of this U.S. patent having beenincorporated by reference hereinabove. Each promoter-primer included aT7 promoter sequence AATTTAATACGACTCACTATAGGGAGA (SEQ ID NO:96) upstreamof an HBV-complementary sequence. Amplification reactions were conductedfor various primer combinations using between about 7 and 33 copies ofthe HBV template, and 1-20 pmoles of each primer in 100 μl of reactionbuffer. Nucleic acids underwent specimen processing and target captureprior to amplification essentially according to the procedures disclosedin published International Patent Application No. PCT/US2000/18685,except that the template was captured using HBV-specificoligonucleotides rather than HIV-specific oligonucleotides.Significantly, all of the capture oligonucleotides used in the procedurewere known to be capable of capturing HBV templates that could beamplified. Accordingly, this procedure focused on the ability of thedifferent primer and probe combinations to cooperate in a multiplexamplification assay. All procedures were conducted using 500 μl aliquotsof stock samples of infected serum containing 5-25 internationalunits/ml (IU/ml) of HBV. Notably, there are approximately 3 copies ofthe HBV genome in one IU of the virion preparation used in thisprocedure. Target nucleic acids and primers were heated to 60° C. for 10minutes and then cooled to 42° C. to facilitate primer annealing.Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (5,600units/reaction) and T7 RNA polymerase (3,500 units/reaction) were thenadded to the mixtures. The final amplification reactions contained 50 mMTris HCl (pH 8.5), 35 mM KCl, 4 mM GTP, 4 mM ATP, 4 mM UTP, 4 mM CTP, 1mM dATP, 1 mM dTTP, 1 mM dCTP, 1 mM dGTP, 20 mM MgCl₂, 20 mMN-Acetyl-L-Cysteine, and 5% (w/v) glycerol. After a one hour incubationat 42° C., the entire 100 μl amplification reaction was subjected to ahybridization assay essentially as described in Example 1. Moreparticularly, one or more of six different probes listed in Table 3 werelabeled with acridinium ester to a specific activity of about 1−1.2×10⁸RLU/pmol and then used in an amount equivalent to 7.5×10⁵ RLU for eachprobe used in the hybridization reaction. To be judged as a positiveresult, the chemiluminescent signal indicating probe hybridization musthave exceeded 50,000 RLU in an assay.

Table 6 presents results from amplification procedures that wereconducted using different combinations of primers, probes and inputlevels of the HBV template. Results in the last column of the table areshown as % positive detection, and additionally indicate the number ofreplicate trials used in the procedure. These results were collectedfrom a number of procedures that were not necessarily carried outcontemporaneously.

TABLE 6 Amplification of HBV Polynucleotide Sequences Using VariousPrimer Combinations HBV-Complementary Sequence(s) HBV of the OppositeStrand genotype A % Positive Promoter-Primer(s) Primer(s) Probe(s)(copies) (# Tested) SEQ ID NO: 24 SEQ ID NO: 5 SEQ ID NO: 58 20 100% SEQ ID NO: 15 (20) SEQ ID NO: 24 SEQ ID NO: 5 SEQ ID NO: 58 20 100%  SEQID NO: 17 SEQ ID NO: 15 (20) SEQ ID NO: 20 SEQ ID NO: 5 SEQ ID NO: 52 2095% SEQ ID NO: 15 SEQ ID NO: 58 (20) SEQ ID NO: 20 SEQ ID NO: 5 SEQ IDNO: 52 20 100%  SEQ ID NO: 24 SEQ ID NO: 15 SEQ ID NO: 58 (20) SEQ IDNO: 25 SEQ ID NO: 5 SEQ ID NO: 58 13 75% SEQ ID NO: 15 (20) SEQ ID NO:25 SEQ ID NO: 5 SEQ ID NO: 58 13 90% SEQ ID NO: 20 SEQ ID NO: 15 (20)SEQ ID NO: 25 SEQ ID NO: 6 SEQ ID NO: 58 13 60% SEQ ID NO: 15 (20) SEQID NO: 25 SEQ ID NO: 6 SEQ ID NO: 58 13 80% SEQ ID NO: 20 SEQ ID NO: 15(20) SEQ ID NO: 25 SEQ ID NO: 7 SEQ ID NO: 58 13 35% SEQ ID NO: 15 (20)SEQ ID NO: 25 SEQ ID NO: 7 SEQ ID NO: 58 13 95% SEQ ID NO: 20 SEQ ID NO:15 (20) SEQ ID NO: 24 SEQ ID NO: 8 SEQ ID NO: 58 20 90% SEQ ID NO: 15(20) SEQ ID NO: 24 SEQ ID NO: 8 SEQ ID NO: 58 20 100%  SEQ ID NO: 17 SEQID NO: 15 (20) SEQ ID NO: 20 SEQ ID NO: 8 SEQ ID NO: 52 20 95% SEQ IDNO: 15 SEQ ID NO: 58 (20) SEQ ID NO: 20 SEQ ID NO: 8 SEQ ID NO: 58 20100%  SEQ ID NO: 24 SEQ ID NO: 15 (20) SEQ ID NO: 25 SEQ ID NO: 8 SEQ IDNO: 58 13 50% SEQ ID NO: 15 (20) SEQ ID NO: 25 SEQ ID NO: 8 SEQ ID NO:58 13 85% SEQ ID NO: 20 SEQ ID NO: 15 (20) SEQ ID NO: 25 SEQ ID NO: 8SEQ ID NO: 58 13 85% SEQ ID NO: 17 SEQ ID NO: 15 (20) SEQ ID NO: 24 SEQID NO: 9 SEQ ID NO: 58 20 95% SEQ ID NO: 15 (20) SEQ ID NO: 24 SEQ IDNO: 9 SEQ ID NO: 58 20 100%  SEQ ID NO: 17 SEQ ID NO: 15 (20) SEQ ID NO:20 SEQ ID NO: 9 SEQ ID NO: 52 20 85% SEQ ID NO: 15 SEQ ID NO: 58 (20)SEQ ID NO: 20 SEQ ID NO: 9 SEQ ID NO: 58 20 100%  SEQ ID NO: 24 SEQ IDNO: 15 (20) SEQ ID NO: 24 SEQ ID NO: 10 SEQ ID NO: 58 20 85% SEQ ID NO:15 (20) SEQ ID NO: 24 SEQ ID NO: 10 SEQ ID NO: 58 20 95% SEQ ID NO: 17SEQ ID NO: 15 (20) SEQ ID NO: 20 SEQ ID NO: 10 SEQ ID NO: 52 20 80% SEQID NO: 15 SEQ ID NO: 58 (20) SEQ ID NO: 20 SEQ ID NO: 10 SEQ ID NO: 5820 95% SEQ ID NO: 24 SEQ ID NO: 15 (20) SEQ ID NO: 20 SEQ ID NO: 11 SEQID NO: 58 13 95% SEQ ID NO: 25 (20) SEQ ID NO: 22 SEQ ID NO: 11 SEQ IDNO: 50 33 70% SEQ ID NO: 57 (10) SEQ ID NO: 23 SEQ ID NO: 11 SEQ ID NO:52 33 85% SEQ ID NO: 58 (20) SEQ ID NO: 22 SEQ ID NO: 11 SEQ ID NO: 5033 70% SEQ ID NO: 23 SEQ ID NO: 57 (10) SEQ ID NO: 23 SEQ ID NO: 12 SEQID NO: 50 20 70% SEQ ID NO: 57 (10) SEQ ID NO: 20 SEQ ID NO: 12 SEQ IDNO: 50 20  0% SEQ ID NO: 57 (10) SEQ ID NO: 17 SEQ ID NO: 12 SEQ ID NO:50 20 60% SEQ ID NO: 57 (10) SEQ ID NO: 23 SEQ ID NO: 12 SEQ ID NO: 5020  0% SEQ ID NO: 20 SEQ ID NO: 57 (20) SEQ ID NO: 23 SEQ ID NO: 12 SEQID NO: 50 20 95% SEQ ID NO: 17 SEQ ID NO: 57 (20) SEQ ID NO: 20 SEQ IDNO: 12 SEQ ID NO: 50 20  0% SEQ ID NO: 17 SEQ ID NO: 57 (20) SEQ ID NO:23 SEQ ID NO: 13 SEQ ID NO: 50 20 90% SEQ ID NO: 57 (10) SEQ ID NO: 20SEQ ID NO: 13 SEQ ID NO: 50 20 20% SEQ ID NO: 57 (10) SEQ ID NO: 17 SEQID NO: 13 SEQ ID NO: 50 20 50% SEQ ID NO: 57 (10) SEQ ID NO: 23 SEQ IDNO: 13 SEQ ID NO: 50 20  0% SEQ ID NO: 20 SEQ ID NO: 57 (20) SEQ ID NO:23 SEQ ID NO: 13 SEQ ID NO: 50 20 10% SEQ ID NO: 17 SEQ ID NO: 57 (20)SEQ ID NO: 20 SEQ ID NO: 13 SEQ ID NO: 50 20  0% SEQ ID NO: 17 SEQ IDNO: 57 (20) SEQ ID NO: 23 SEQ ID NO: 14 SEQ ID NO: 50 20 80% SEQ ID NO:57 (10) SEQ ID NO: 20 SEQ ID NO: 14 SEQ ID NO: 50 20 70% SEQ ID NO: 57(10) SEQ ID NO: 17 SEQ ID NO: 14 SEQ ID NO: 50 20 70% SEQ ID NO: 57 (10)SEQ ID NO: 23 SEQ ID NO: 14 SEQ ID NO: 50 20 95% SEQ ID NO: 20 SEQ IDNO: 57 (20) SEQ ID NO: 23 SEQ ID NO: 14 SEQ ID NO: 50 20 10% SEQ ID NO:17 SEQ ID NO: 57 (20) SEQ ID NO: 20 SEQ ID NO: 14 SEQ ID NO: 50 20  0%SEQ ID NO: 17 SEQ ID NO: 57 (20) SEQ ID NO: 23 SEQ ID NO: 15 SEQ ID NO:50 20 90% SEQ ID NO: 57 (10) SEQ ID NO: 20 SEQ ID NO: 15 SEQ ID NO: 5020 80% SEQ ID NO: 57 (10) SEQ ID NO: 17 SEQ ID NO: 15 SEQ ID NO: 50 2040% SEQ ID NO: 57 (10) SEQ ID NO: 23 SEQ ID NO: 15 SEQ ID NO: 50 20 45%SEQ ID NO: 20 SEQ ID NO: 57 (20) SEQ ID NO: 23 SEQ ID NO: 15 SEQ ID NO:50 20 80% SEQ ID NO: 17 SEQ ID NO: 57 (20) SEQ ID NO: 20 SEQ ID NO: 15SEQ ID NO: 50 20  0% SEQ ID NO: 17 SEQ ID NO: 57 (20) SEQ ID NO: 24 SEQID NO: 15 SEQ ID NO: 52 20 100%  SEQ ID NO: 58 (10) SEQ ID NO: 25 SEQ IDNO: 15 SEQ ID NO: 52 20 100%  SEQ ID NO: 58 (10) SEQ ID NO: 27 SEQ IDNO: 15 SEQ ID NO: 52 20 100%  SEQ ID NO: 58 (10) SEQ ID NO: 28 SEQ IDNO: 15 SEQ ID NO: 52 20 100%  SEQ ID NO: 58 (10) SEQ ID NO: 24 SEQ IDNO: 15 SEQ ID NO: 52 13 80% SEQ ID NO: 58 (10) SEQ ID NO: 25 SEQ ID NO:15 SEQ ID NO: 52 13 100%  SEQ ID NO: 58 (10) SEQ ID NO: 27 SEQ ID NO: 15SEQ ID NO: 52 13 100%  SEQ ID NO: 58 (10) SEQ ID NO: 28 SEQ ID NO: 15SEQ ID NO: 52 13 100%  SEQ ID NO: 58 (10) SEQ ID NO: 22 SEQ ID NO: 15SEQ ID NO: 50 33 90% SEQ ID NO: 57 (10) SEQ ID NO: 22 SEQ ID NO: 15 SEQID NO: 50 33 100%  SEQ ID NO: 23 SEQ ID NO: 57 (10) SEQ ID NO: 23 SEQ IDNO: 15 SEQ ID NO: 54 20 100%  SEQ ID NO: 26 SEQ ID NO: 11 (30) SEQ IDNO: 23 SEQ ID NO: 15 SEQ ID NO: 54  7 85% SEQ ID NO: 26 SEQ ID NO: 11(20) SEQ ID NO: 21 SEQ ID NO: 15 SEQ ID NO: 50 33 100%  SEQ ID NO: 11SEQ ID NO: 57 (20) SEQ ID NO: 29 SEQ ID NO: 15 SEQ ID NO: 54 20 90% (20)SEQ ID NO: 29 SEQ ID NO: 11 SEQ ID NO: 54 20 90% (20) SEQ ID NO: 30 SEQID NO: 15 SEQ ID NO: 54 20 100%  (20) SEQ ID NO: 30 SEQ ID NO: 11 SEQ IDNO: 54 20 100%  (20) SEQ ID NO: 31 SEQ ID NO: 15 SEQ ID NO: 54 20 95%(20) SEQ ID NO: 31 SEQ ID NO: 11 SEQ ID NO: 54 20 95% (20) SEQ ID NO: 32SEQ ID NO: 15 SEQ ID NO: 54 20 95% (20) SEQ ID NO: 32 SEQ ID NO: 11 SEQID NO: 54 20 95% (20) SEQ ID NO: 16 SEQ ID NO: 15 SEQ ID NO: 52 50 60%SEQ ID NO: 58 M13 Template (10) SEQ ID NO: 18 SEQ ID NO: 15 SEQ ID NO:52 50 70% SEQ ID NO: 58 M13 Template (10) SEQ ID NO: 19 SEQ ID NO: 15SEQ ID NO: 52 50 90% SEQ ID NO: 58 M13 Template (10)

The results presented in Table 6 showed that many of the primercombinations gave very high levels of HBV detectability, even attemplate levels as low as 7 copies/reaction. Significantly, resultsindicating that HBV templates could be detected in these assays,demonstrated that the relevant primer combinations could cooperate toamplify HBV nucleic acid sequences, and further demonstrated that otherconstituents of the HIV/HCV multiplex reaction did not interfere withthe ability of the HBV primer combinations to function in the complexreaction mixture. Positive results, meaning that a non-zero level of HBVdetectability was observed in a trial, indicated that a combination ofprimers was useful in an assay that amplified HBV only, as well as in amultiplex assay that was capable of amplifying HIV-1 and HCV templates.Negative results that indicated amplification and detection of HBVsequences did not take place were relevant only to the multiplex assay.Negative results may have been due, for example, to an undesirableinteraction between the HBV primers and the primers used for amplifyingone of the other viral targets in the multiplex reaction. As supportedby the sensitivity data appearing below, primer combinations that gavelow, but measurable levels of HBV detectability in the results presentedherein indicated successful amplification of HBV templates andestablished the combination as a useful component of an HBV nucleic acidamplification assay. Other primers contained within the upstream anddownstream domains defined by these useful primers also can be used foramplifying HBV sequences.

The results presented in Table 6 further indicated that promoter-primerswhich included the HBV-complementary sequences of SEQ ID NO:16, SEQ IDNO:18, or SEQ ID NO:19 participated in amplification reactions that gavegood levels of detectability, even when the reactions included only asingle promoter-primer and when a single-stranded M13 clone was thesource of HBV genotype A templates. This result differed somewhat fromthe apparent requirement for two promoter-primers when a single-strandedM13 clone served as the source of HBV genotype G templates, as indicatedby the results appearing in Table 9. While not wishing to be bound byany particular theory, the comparatively good results observed in onlysome instances when single promoter-primers were used in conjunctionwith single-stranded M13 templates may have reflected different levelsof assay sensitivity.

In certain preferred embodiments, a set of at least two primers foramplifying HBV nucleic acid is provided which includes: (i) a firstamplification primer comprising an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:22 or SEQID NO:23; and (ii) a second amplification primer comprising anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:15 or SEQ ID NO:11. Optional sequences that arenon-complementary to the HBV target sequence, such as a promotersequence that is not present in the HBV genome, may be appended to theprimers at positions located upstream of the target-complementarysequences. In a particularly preferred combination, the firstamplification primer is a promoter-primer that comprises anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:23 (e.g., SEQ ID NO:40), and the secondamplification primer comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:11.

As in the previous Example, procedures that identified useful captureoligonucleotides also employed stocks of HBV genotype A-positive plasmaas the source of HBV nucleic acid. Each oligonucleotide that underwenttesting included an HBV-specific sequence linked to an oligo-(dA) tailsequence. When combined with the HBV target and magnetic particles thatdisplayed oligo-(dT), functional capture oligonucleotides bridged theHBV DNA and the particle, thereby immobilizing the HBV target. Removingthe particulate complexes from solution represented a means forenriching the HBV template. In the procedure described below, captureoligonucleotides were contacted with the HBV DNA and magnetic particlesmodified with oligo-(dT). Collected particles were washed, the bound HBVsequences amplified in an in vitro nucleic acid amplification reaction,and the resulting amplification products detected in homogenousprotection assays. Positive results indicated that the captureoligonucleotide had immobilized the HBV DNA target onto the magneticparticle during the target capture step, and that the amplification anddetection steps were successful.

The following Example describes the methods used for testing candidateHBV capture oligonucleotides. In addition to the HBV-specific targetcapture, amplification primer, and probes described in this procedure,the reactions also included the above-described oligonucleotidesspecific for the HIV-1 and HCV analytes. The HIV-1 and HCVoligonucleotides did not substantially contribute to detection of theHBV analyte.

EXAMPLE 3 Detection of HBV Target Sequences Using Different CaptureOligonucleotides

HBV-infected plasma samples having volumes of 500 μl and containing 6-20copies of the HBV genome were dispersed in 400 μl of lysis/capturereagent containing a total of 1.6 pmoles of capture oligonucleotide(either 1.6 pmoles of a single capture oligonucleotide, 0.8 pmoles ofeach of two capture oligonucleotides, or 0.53 pmoles of each of threecapture oligonucleotides) and about 40 μg of 0.7-1.05 p paramagneticparticles (Seradyn, Indianapolis, Ind.) covalently linked topoly-(dT₁₄). Capture oligonucleotides used in the procedure had thesequences given in Table 4. The lysis/capture reagent further includedan HIV-1 internal amplification control template, HIV-1 and HCV-specificcapture oligonucleotides, and a 100 mM HEPES-buffered solutioncontaining 294 mM lithium lauryl sulfate, 730 mM lithium chloride, and50 mM lithium hydroxide. As stated above, a 5′-TTT-3′ spacer sequencewas interposed between the HBV-complementary sequence and the oligo-(dA)tail region for each of the capture oligonucleotides shown in Table 4,except for those that included the sequences of SEQ ID NO:71 and SEQ IDNO:80. Notably, testing of some capture oligonucleotides havingidentical sequences except for the presence or absence of the 5′-TTT-3′sequence gave substantially similar results, thereby indicating that thespacer sequence was optional. The mixtures were heated to 55-60° C. forabout 15-30 minutes, and then cooled to room temperature to allowhybridization. A magnetic field was applied to collect the particlecomplexes containing the immobilized capture oligonucleotide and HBV DNAusing procedures such as those described by Wang in U.S. Pat. No.4,895,650. The particles were washed twice with 1 ml of a washing buffer(10 mM HEPES, 6.5 mM NaOH, 1 mM EDTA, 0.3% (v/v) ethanol, 0.02% (w/v)methyl-paraben, 0.01% (w/v) propyl-paraben, 150 mM NaCl, 0.1% (w/v)sodium lauryl sulfate). Washed particles were then resuspended in 75 p.1 of the amplification reagent described under Example 2. This reagentincluded salts, nucleotides, ribonucleotides, HBV-specific primers, aswell as primers capable of amplifying HIV-1 and HCV target sequences.The HBV target nucleic acid was then amplified, and the amplificationproducts detected using a homogenous protection assay, essentially asdescribed under Example 1. Reactions that gave positive signals whenhybridized with a probe specific for the internal control amplicon, orwith a probe specific for the HBV amplicon, were scored as valid runs.In order for a valid run to be considered positive for the presence ofHBV amplicons, the chemiluminescent signal indicating probehybridization must have exceeded 50,000 RLU in an assay.

Table 7 presents sample results correlating the identity of theHBV-specific capture oligonucleotide(s) and the ability of the system toamplify and detect HBV sequences efficiently. To achieve a positiveresult in the amplification reactions, the HBV capture oligonucleotidemust have been able to act cooperatively with the amplification primersand probe(s) to capture HBV template nucleic acids, amplify the HBVtemplate nucleic acids, and then detect the amplified nucleic acids.However, the experimental design also required that primers used foramplifying HIV-1 and HCV target nucleic acids would not substantiallyinterfere with the capture, amplification and detection of HBV templatenucleic acids. Thus, positive results identified combinations of captureoligonucleotides, amplification primers and detection probes that werefunctional not only in an HBV-specific assay, but also in the context ofa multiplex reaction capable of amplifying HIV-1 and HCV target nucleicacids.

Notably, promoter-primers used in this procedure and listed in Table 7are identified by the complete sequence that included the T7 promoter.It is to be understood however, that the HBV-complementary portions ofthe promoter-primers represent essential sequences for performingamplification reactions by alternative protocols, such as the polymerasechain reaction, with the promoter sequence being optional. Thus, it isto be understood that SEQ ID NOs:33-49 possess optional promotersequences, and that the corresponding SEQ ID NOs:16-32 represent therespective essential HBV-complementary sequences. These latterHBV-complementary sequences are useful in conjunction with oppositestrand primers for amplifying HBV nucleic acids.

TABLE 7 Efficiency of HBV Detection Using Different Combinations ofCapture Oligonucleotides, Amplification Primers and Detection ProbesCapture Amplification Detection Template Copy % PositiveOligonucleotide(s) Primers Probe(s) No. (# Valid Runs) SEQ ID NO: 70 SEQID NO: 39 SEQ ID NO: 50 6.7 53% SEQ ID NO: 80 SEQ ID NO: 40 SEQ ID NO:57 (40) SEQ ID NO: 15 SEQ ID NO: 11 SEQ ID NO: 70 SEQ ID NO: 40 SEQ IDNO: 52 20 85% SEQ ID NO: 86 SEQ ID NO: 15 SEQ ID NO: 58 (80) SEQ ID NO:71 SEQ ID NO: 40 SEQ ID NO: 50 20 30% SEQ ID NO: 15 SEQ ID NO: 57 (20)SEQ ID NO: 71 SEQ ID NO: 39 SEQ ID NO: 50 6 33% SEQ ID NO: 78 SEQ ID NO:40 SEQ ID NO: 57 (18) SEQ ID NO: 15 SEQ ID NO: 11 SEQ ID NO: 71 SEQ IDNO: 40 SEQ ID NO: 50 20 95% SEQ ID NO: 80 SEQ ID NO: 15 SEQ ID NO: 57(20) SEQ ID NO: 71 SEQ ID NO: 39 SEQ ID NO: 50 12 60% SEQ ID NO: 80 SEQID NO: 40 SEQ ID NO: 57 (20) SEQ ID NO: 15 SEQ ID NO: 11 SEQ ID NO: 71SEQ ID NO: 40 SEQ ID NO: 50 6.7 32% SEQ ID NO: 80 SEQ ID NO: 15 SEQ IDNO: 57 (19) SEQ ID NO: 71 SEQ ID NO: 40 SEQ ID NO: 50 6.7 31% SEQ ID NO:82 SEQ ID NO: 15 SEQ ID NO: 57 (45) SEQ ID NO: 71 SEQ ID NO: 40 SEQ IDNO: 52 20 84% SEQ ID NO: 86 SEQ ID NO: 15 SEQ ID NO: 58 (80) SEQ ID NO:71 SEQ ID NO: 39 SEQ ID NO: 50 12 90% SEQ ID NO: 86 SEQ ID NO: 40 SEQ IDNO: 57 (20) SEQ ID NO: 15 SEQ ID NO: 11 SEQ ID NO: 72 SEQ ID NO: 40 SEQID NO: 54 20 62% SEQ ID NO: 15 (60) SEQ ID NO: 43 SEQ ID NO: 11 SEQ IDNO: 72 SEQ ID NO: 40 SEQ ID NO: 54 20 98% SEQ ID NO: 80 SEQ ID NO: 15(60) SEQ ID NO: 87 SEQ ID NO: 43 SEQ ID NO: 11 SEQ ID NO: 73 SEQ ID NO:40 SEQ ID NO: 52 20 36% SEQ ID NO: 15 SEQ ID NO: 58 (45) SEQ ID NO: 73SEQ ID NO: 40 SEQ ID NO: 52 20 84% SEQ ID NO: 80 SEQ ID NO: 15 SEQ IDNO: 58 (45) SEQ ID NO: 73 SEQ ID NO: 40 SEQ ID NO: 54 20 100%  SEQ IDNO: 80 SEQ ID NO: 15 (60) SEQ ID NO: 87 SEQ ID NO: 43 SEQ ID NO: 11 SEQID NO: 73 SEQ ID NO: 40 SEQ ID NO: 54 6.7 70% SEQ ID NO: 80 SEQ ID NO:15 (60) SEQ ID NO: 87 SEQ ID NO: 43 SEQ ID NO: 11 SEQ ID NO: 74 SEQ IDNO: 40 SEQ ID NO: 50 20 50% SEQ ID NO: 15 SEQ ID NO: 57 (20) SEQ ID NO:74 SEQ ID NO: 40 SEQ ID NO: 50 6.7  5% SEQ ID NO: 15 SEQ ID NO: 57 (20)SEQ ID NO: 74 SEQ ID NO: 40 SEQ ID NO: 52 20 88% SEQ ID NO: 85 SEQ IDNO: 15 SEQ ID NO: 58 (80) SEQ ID NO: 74 SEQ ID NO: 40 SEQ ID NO: 50 20100%  SEQ ID NO: 86 SEQ ID NO: 15 SEQ ID NO: 57 (20) SEQ ID NO: 76 SEQID NO: 40 SEQ ID NO: 52 20 60% SEQ ID NO: 15 SEQ ID NO: 58 (45) SEQ IDNO: 77 SEQ ID NO: 40 SEQ ID NO: 50 20 35% SEQ ID NO: 15 SEQ ID NO: 57(20) SEQ ID NO: 78 SEQ ID NO: 40 SEQ ID NO: 52 20 88% SEQ ID NO: 86 SEQID NO: 15 SEQ ID NO: 58 (77) SEQ ID NO: 80 SEQ ID NO: 40 SEQ ID NO: 5020 26% SEQ ID NO: 15 SEQ ID NO: 57 (19) SEQ ID NO: 80 SEQ ID NO: 40 SEQID NO: 52 20 74% SEQ ID NO: 87 SEQ ID NO: 15 SEQ ID NO: 58 (90) SEQ IDNO: 81 SEQ ID NO: 40 SEQ ID NO: 52 20 79% SEQ ID NO: 86 SEQ ID NO: 15SEQ ID NO: 58 (80) SEQ ID NO: 82 SEQ ID NO: 40 SEQ ID NO: 50 20 60% SEQID NO: 15 SEQ ID NO: 57 (20) SEQ ID NO: 84 SEQ ID NO: 40 SEQ ID NO: 5020 40% SEQ ID NO: 15 SEQ ID NO: 57 (20) SEQ ID NO: 75 SEQ ID NO: 40 SEQID NO: 52 20 58% SEQ ID NO: 15 SEQ ID NO: 58 (45) SEQ ID NO: 79 SEQ IDNO: 40 SEQ ID NO: 52 20 81% SEQ ID NO: 86 SEQ ID NO: 15 SEQ ID NO: 58(80) SEQ ID NO: 83 SEQ ID NO: 40 SEQ ID NO: 52 20 74% SEQ ID NO: 86 SEQID NO: 15 SEQ ID NO: 58 (77)

The results presented in Table 7 confirmed that certain combinations ofcapture oligonucleotides, amplification primers and detection probesgave results that were substantially better than others. For example,the promoter-primer containing the HBV-complementary sequence of SEQ IDNO:23 (i.e., SEQ ID NO:40) and opposite strand primer of SEQ ID NO:15were capable of producing positive results in 100% of amplificationreactions containing only 20 copies of the HBV template. These primersrepresent a particularly preferred combination for amplifying HBVnucleic acids. Similarly, the above-described probes of SEQ ID NO:54,and the combination of the probes of SEQ ID NO:50 and SEQ ID NO:57 werecapable of detecting the HBV amplicons in a highly efficient manner.Probes having these sequences, or probes having sequences complementarythereto, are preferred for detecting HBV nucleic acids.

The results presented in Table 7 further showed that certaincombinations of capture oligonucleotides, amplification primers anddetection probes worked synergistically in the multiplex amplificationsystem. For example, the results shown for the trial conducted usingcapture oligonucleotides of SEQ ID NO:74 and SEQ ID NO:86, theamplification primer of SEQ ID NO:15 and the promoter-primer containingthe HEY-complementary sequence of SEQ ID NO:23 (i.e., SEQ ID NO:40),together with the probes of SEQ ID NO:50 and SEQ ID NO:57 using 20copies of the HBV template nucleic acid demonstrated that each of theseprimers and probes was capable of amplifying and detecting HBV templatenucleic acids in 100% of the valid runs. When the same primers andprobes were used in combination with only one of the captureoligonucleotides identified by SEQ ID NO:71 and SEQ ID NO:80 inreactions containing 20 copies of the HBV template nucleic acid,positive results were achieved in only about 30% of the valid runs.However, when the capture oligonucleotides that included the sequencesof SEQ ID NO:71 and SEQ ID NO:80 were used in combination with eachother, with each oligonucleotide being present in half of the totalamount used in the trials having only a single capture oligonucleotide,dramatically higher results approaching 100% were achieved. Thisdemonstrated how certain combinations of the assay reagents gaveunexpectedly good results, even at very low levels of input target.

Example 4 describes a procedure that used a particular set of theinvented oligonucleotides to illustrate how the different amplificationassays described herein were characterized by different sensitivityranges. The findings from this procedure established that 100% positiveHBV detection results could be obtained in an amplified assay when thelevel of template used in the amplification reaction exceeded a certainthreshold value. As will be apparent from comparing the below-presentedresults with those from the other Examples herein, this threshold valuedepended on the particular combination of oligonucleotides used in theamplification reaction. Notably, the assay described in this Exampledetected HBV genotypes A-G, even when the probe of SEQ ID NO:57 was theonly probe used in the detection step.

EXAMPLE 4 Detection of HBV Nucleic Acids

Nucleic acid amplification reactions were conducted essentially asdescribed in the preceding Examples, except that primers necessary foramplifying HIV-1 and HCV sequences were omitted. Captureoligonucleotides used in the procedure included the HBV-complementarysequences of SEQ ID NO:71 and SEQ ID NO:80. Amplification primersincluded the sequences of SEQ ID NO:22 and SEQ ID NO:23, each of thesesequences having a T7 promoter sequence appended at its 5′-end. Thecomplete sequences of the promoter primers were given by SEQ ID NO:39,and SEQ ID NO:40. Opposite strand primers had sequences given by SEQ IDNO:15 and SEQ ID NO:11. Acridinium ester-labeled probes used in theprocedure had sequences given by SEQ ID NO:50 and SEQ ID NO:57, asdescribed above. 500 μl aliquots of virion-containing plasma weresubjected to the above-described detergent lysis and target captureprocedure. Amplification reactions were carried out, and theamplification products detected, also using the above-describedprocedures. Virion concentrations of samples processed in this procedureranged from 0-100 EU/ml, where 1 EU corresponds to approximately 3copies of the HBV genome. Positive detection of HBV target sequences inthe amplification reactions was assessed essentially as described in thepreceding Examples.

The results presented in Table 8 indicated that 100% of the reactionsconducted using 300 copies of the HBV template gave positive results,and that 95% positively was achieved at some level between 75 and 150copies per reaction. Significantly, substantially similar results wereobtained when the amplification reactions were conducted in the absenceof the primer identified as SEQ ID NO:15. Thus, a preferred combinationof primers for amplifying HBV nucleic acids has HBV-complementarysequences given by SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:11, withthe primer of SEQ ID NO:15 being optional. A preferred probe compositionfor detecting HBV amplification products includes the probe of SEQ IDNO:57, with further inclusion of the probe of SEQ ID NO:50 beingoptional.

TABLE 8 Sensitivity Testing Establishes a Range for the Level ofTemplates Detected in an Assay HBV Genotype A (copies) % Positive 300100.0 150 99.0 75 91.7 36 71.0 18 43.9 0 0.3

The results in Table 8 further confirmed that even primer combinationslisted in Tables 6 and 7 that did not result in high levels of HBVdetection at the specified level of input template were capable ofidentifying HBV-containing samples at levels approaching 100%detectability when the amount of template was sufficiently high. Basedon these findings, it was concluded that primer combinations that gaveat least some measure of HBV detection when the amount of input templateranged from 6-20 copies per reaction were deemed useful in proceduresfor amplifying HBV nucleic acids in vitro. While it may be desirable insome circumstances to use combinations of primers that result in highlysensitive assays, other circumstances may benefit from using assayscharacterized by somewhat lower sensitivities. Thus, any of the primercombinations disclosed herein that gave measurable results in Tables 6and 7 can be used as components of HBV nucleic acid amplificationassays, and fall within the scope of the invention.

Example 5 illustrates how the qualitative assay from the precedingExample can be adapted to a quantitative format. Spike recovery of threeHBV genotype A positive controls provided a quantitative assessment ofassay performance. The assay relied on the use of a pseudo target inaccordance with U.S. Pat. No. 6,294,338, the disclosure of which ishereby incorporated by reference. Although either RNA or DNA pseudotargets can be used in quantitative assays, the following procedure wasconducted using an RNA pseudo target.

EXAMPLE 5 Quantitative Assay for Measuring HBV

The oligonucleotide components used in the quantitative assay wereidentical to the reagents used in the preceding Example, except that theprobe of SEQ ID NO:50 was omitted. Each amplification reactionadditionally included an RNA pseudo target having the sequence of SEQ IDNO:97 in an amount corresponding to about 80×10⁶ copies. HBV genotype Acalibrators having concentrations ranging from 380-12,000 HBV IU/ml wereused as the source of HBV templates in separate reactions to producestandard curves. TMA reactions were conducted for a period of 60minutes, substantially as described above. Detection of amplified HBVsequences was carried out using a standard homogeneous protection assaythat employed the above-described probe of SEQ ID NO:57 as a combinationof acridinium ester-labeled and unlabeled species in a ratio of 1:6.25.Use of the unlabeled probe allowed all chemiluminescent signals to fallwithin the reliable response range of the luminometer that measuredspecifically hybridized probe. Pseudo target amplicons were not detectedby the probe that was used in this procedure.

The results presented in FIG. 2 showed how the quantitative assay gave asubstantially linear relationship between the amount of input HBVtemplate and the hybridization signal strength over a broad range of HBVconcentrations that extended from 0-12,000 IU/ml. The lower limit ofdetection in this assay was about 1 IU/ml, or between 1 and 2 copies ofthe HBV nucleic acid in a single amplification reaction. Thus, thisquantitative assay was highly sensitive and had the ability to measureHBV concentrations, or template copy numbers, over a very broad range ina precise manner.

Example 6 describes a procedure which demonstrated how the use of asecond promoter-primer in a TMA reaction could positively influencedetectability of different HBV genotypes. In addition to theHBV-specific target capture oligonucleotides, amplification primer, andprobes described in this procedure, the reactions also included theabove-described oligonucleotides specific for the HIV-1 and HCVanalytes. The HIV-1 and HCV oligonucleotides did not substantiallycontribute to detection of the HBV analyte.

EXAMPLE 6 Improved Detectability of HBV Genotypes

Genotyped HBV-containing samples (genotypes A-F) were obtained fromMillenium Biotech, Inc., (Ft. Lauderdale, Fla.) or Boston Biomedica(West Bridgewater, Mass.). Quantitation values were supplied by thevendor's certificate of analysis and were based on results from theAMPLICOR HBV MONITOR assay (Roche Diagnostics Corp.). In the absence ofplasma samples containing HBV genotype G virions, a single-stranded M13clone that included HBV genotype G nucleic acid sequences correspondingto all of the relevant oligonucleotides, except for captureoligonucleotide that included SEQ ID NO:87, served as the template forprocedures that amplified sequences of this genotype. Panel members weremade by serial dilution of patient plasma samples using negative humanplasma. 400 μl of target capture reagent (HEPES-buffered detergentsolution containing the above-described capture oligonucleotides havingthe HBV-complementary sequences of SEQ ID NO:73, SEQ ID NO:80 and SEQ IDNO:87, together with magnetic particles) containing internal control and500 μl of each specimen or panel member were pipetted manually into TenTube Units (TTUs). After addition of the target capture reagent andspecimen, the TTUs were vortexed and incubated in a 60° C. water bathfor 20 minutes, followed by room temperature incubation (15-30° C.) for14-20 minutes. The rack of TTUs was then placed in a magnetic separationbay for 9-20 minutes. Liquid was aspirated from each tube and thenreplaced with 1 ml of wash solution. The rack was vortexed and againplaced on the separation bay for 4-10 minutes. The wash solution wasaspirated from each tube and wash and separation steps were repeated,finishing with a final aspiration. After the final aspiration step, 75μl of amplification reagent (including one of two different primerformulations, dNTPs, NTPs and cofactors in Tris-buffered solution) wasadded to each tube. Primer formulation A included a promoter-primerhaving the HBV-complementary sequence of SEQ ID NO:23 (i.e., SEQ IDNO:40), as well as opposite strand primers identified by SEQ ID NO:15and SEQ ID NO:11. Primer formulation B additionally included apromoter-primer having the HBV-complementary sequence of SEQ ID NO:26(i.e., SEQ ID NO:43). Each mixture was overlaid with 200 μl of inert oilto prevent evaporation during the amplification step, and the rack ofTTUs vortexed to resuspend the microparticles. Prior to the addition of25 μl of enzyme reagent, (MMLV reverse transcriptase and T7 RNApolymerase in HEPES/Tris-buffered solution), the rack was incubated in awater bath at 60° C. for 10 minutes, followed by equilibration at 41.5°C. for 9-20 minutes. Immediately after adding the enzyme reagent, therack of TTUs was removed from the incubator and shaken to mix. The rackwas then incubated in the water bath at 41.5° C. for 60 minutes. Afteramplification, 100 μl of probe reagent, which included theabove-described acridinium ester labeled probes of SEQ ID NO:54 insuccinate buffered detergent solution, was added to each tube, vortexed,and incubated in a water bath at 60° C. for 15 minutes. Following thecompletion of the probe hybridization step, 250 μl of selection reagent(borate-buffered solution with surfactant) was added to each tube. Tubeswere vortexed, and then incubated at for 10 minutes. After removal fromthe 60° C. water bath, the rack of TTUs was cooled in a water bath at19-27° C. for 10-75 minutes and then placed in a LEADER HC+ luminometer(Gen-Probe Incorporated; San Diego, Calif.) configured for automaticinjection of 200 μl of a solution of 0.1% hydrogen peroxide and 1 mMnitric acid; and 200 μl of 1 N NaOH. To determine reactivity, theresulting chemiluminescence was compared to a cutoff value generatedfrom the positive and negative calibrators that had been included ineach 100 tube run. To monitor assay performance for each specimenreaction, an internal control contained in the target capture reagentwas added to each test specimen. The internal control consisted of an invitro synthesized transcript containing a portion of HIV-1 and a uniquesequence targeted by the internal control probe. The internal controlsignal in each reaction was discriminated from the HBV signal by thedifferential kinetics of light emission from probes with differentlabels. The internal control amplification product was detected using aprobe with rapid emission of light while the amplicon specific to HBVwas detected using probes with slower kinetics of light emission.Software receiving inputs from the luminometer differentiated betweenthe two signals. Results of these procedures are shown in Table 9.

TABLE 9 Improved Detectability of HBV Genotypes HBV Copies/ PrimerFormulation A Primer Formulation B Genotype Reaction (% Positive) (%Positive) A 50 100 100 15 100 100 A 50 100 100 15 100 100 B 50 85 100 1560 80 C 50 95 100 15 83 100 D 50 95 100 15 60 95 E 50 100 100 15 70 100F 50 100 100 15 100 100 G 50 0 100 15 0 85

As indicated by the results presented in Table 9, the primer formulationthat included two promoter-primers advantageously detected HBV in 100%of the samples containing each of the different HBV genotypes at the 50copy level. This was not true when the HBV genotype B, C, D, E or Gtemplates were used at the same template copy level in the amplificationreactions that included only one promoter-primer. The negative resultsobtained for TMA reactions conducted using a single promoter-primer andthe cloned HBV genotype G template were artifacts due to the use of atemplate that was entirely single-stranded. Separate testing performedusing HBV genotype G virions as the source of templates, and primerformulation B as the source of primers, gave 100% detectability at both15 and 50 copies/reaction. When it is desirable to amplify the nucleicacids of HBV genotypes A-G in a highly sensitive manner, it is preferredto use two first strand primers and at least one second strand primer inthe reaction. In a particular instance, when it is desirable to amplifythe nucleic acids of HBV genotypes A-G in a highly sensitive TMAreaction, it is preferred to use two promoter primers and at least onenon-promoter primer in the reaction. Of course, in vitro nucleic acidamplification reactions based on thermal cycling protocols, orprocedures that involve a step for physically separating newlysynthesized strands of nucleic acid from their templates, can beconducted using only one upstream primer and one downstream primer.

The following Examples present evidence showing that combinations of theoligonucleotides disclosed herein could be used in multiplex reactionsthat were also capable of detecting each of HIV-1 and HCV nucleic acidsin a highly sensitive manner.

Example 7 describes a procedure wherein HIV-1 target nucleic acids weredetected using the oligonucleotides specific for HIV-1 and theamplification and detection protocols disclosed herein.

EXAMPLE 7 Detection of HIV-1 in a Multiplex Amplification Reaction

Samples of the HIV-1 WHO Standard, Subtype B (97/656) were diluted intonegative human plasma to give viral titers ranging from 0-300 IU/ml. 500μA samples of this dilution panel were then subjected to specimenprocessing, target capture, amplification and detection according to theprocedures described under Example 2 using the HIV-1 specificoligonucleotides of SEQ ID NOs:100-114 together with the HCV specificoligonucleotides of SEQ ID NOs:115-125 and HBV specificoligonucleotides. The HIV-1 specific capture oligonucleotides had thesequences of SEQ ID NOs:113-114. The HIV-1 specific promoter-primers ofSEQ ID NOs:100-101 had the essential HIV-complementary sequences of SEQID NOs:98-99 respectively appended to an upstream promoter sequence.Opposite strand primers had the sequences of SEQ ID NOs:102-103. Probeshad the sequences of SEQ ID NOs:106-108. The HCV specific captureoligonucleotides had the sequences of SEQ ID NOs:124-125. The HCVspecific promoter-primer of SEQ ID NO:116 had the essentialHCV-complementary sequence of SEQ ID NO:115 appended to an upstreampromoter sequence. Opposite strand primers had the sequences of SEQ IDNOs:117-118. Probes had the sequences of SEQ ID NOs:120 and 122. HBVspecific oligonucleotides in this procedure included the captureoligonucleotides having the HBV-complementary sequences of SEQ ID NO:73,SEQ ID NO:80 and SEQ ID NO:87; amplification primers of SEQ ID NO:40,SEQ ID NO:43, SEQ ID NO:15 and SEQ ID NO:11; and the above-describedhybridization detection probes of SEQ ID NO:54. The HBV specificpromoter-primers of SEQ ID NO:40 and 43 respectively had the essentialHBV-complementary sequences of SEQ ID NO:23 and 26 appended to anupstream promoter sequence. Twenty replicates were carried out, and the95% detection probability at the 95% confidence interval determined.Results of the procedure are presented in Table 10.

TABLE 10 Detection of HIV-1 Nucleic Acids in Multiplex AmplificationReaction Titer 95% Detection (IU/ml) % Positive Probability 300 100 13.0100 100 (10.1 to 19.5) 33.3 100 11.1 84 3.7 53 1.23 21 0 0

The results shown in Table 10 clearly indicated that HIV-1 nucleic acidswere detected in the multiplex reaction that was also capable ofdetecting HCV and HBV nucleic acids. Moreover, additional testingindicated that HIV-1 type/group detectability in this assay included A,B, C, D, E, F, G, Group N and Group O at levels approaching 100%positively when reactions contained 100 copies/ml or more of HIV-1. Asindicated hereinabove, the collection of HIV-1 specific oligonucleotidesused in this procedure can be used in stand-alone assays for detectingHIV-1, or in combination with the oligonucleotides specific for HCV, oralternatively HBV, to create duplex assays.

Example 8 describes a procedure wherein HCV target nucleic acids weredetected using the oligonucleotides specific for HCV and theamplification and detection protocols disclosed herein.

EXAMPLE 8 Detection of HCV in a Multiplex Amplification Reaction

Samples of the HCV WHO Standard, genotype 1 (96/790) were diluted intonegative human plasma to give viral titers ranging from 0-100 IU/ml. 500μl samples of this dilution panel were then subjected to specimenprocessing, target capture, amplification and detection using theoligonucleotides and procedures described under the previous Example.Twenty replicates were carried out, and the 95% detection probability atthe 95% confidence interval determined. Results of the procedure arepresented in Table 11.

TABLE 11 Detection of HCV Nucleic Acids in Multiplex AmplificationReaction Titer 95% Detection (IU/ml) % Positive Probability 100 100 1.633.3 100 (1.2 to 2.3) 11.1 100 3.7 100 1.23 80 0.41 47 0 0

The results shown in Table 11 clearly indicated that HCV nucleic acidswere detected in the multiplex reaction that was also capable ofdetecting HIV-1 and HBV nucleic acids. Moreover, additional testingindicated that HCV genotypes 1, 1b, 2b, 2a/c, 3, 3e, 4, 4a, 4b/c, 5, 5a,6, 6a were all detected with 100% positively when reactions contained100 copies/ml or more of HCV. As indicated hereinabove, the collectionof HCV specific oligonucleotides used in this procedure can be used instand-alone assays for detecting HCV, or in combination with theoligonucleotides specific for HIV, or alternatively HBV, to createduplex assays.

Example 9 describes a procedure wherein HBV target nucleic acids weredetected using the oligonucleotides specific for HBV and theamplification and detection protocols disclosed herein. The experimentalresults presented below quantitatively illustrate the sensitivity ofthis assay.

EXAMPLE 9 Detection of HBV in a Multiplex Amplification Reaction

Samples of the HBV WHO Standard, genotype A (97/746) were diluted intonegative human plasma to give viral titers ranging from 0-45 IU/ml. 500μl samples of this dilution panel were then subjected to specimenprocessing, target capture, amplification and detection using theoligonucleotides and procedures described under Example 7. Eightyreplicates were carried out, and the 95% detection probability at the95% confidence interval determined. Results of the procedure arepresented in Table 12.

TABLE 12 Detection of HBV Nucleic Acids in Multiplex AmplificationReaction Titer 95% Detection (IU/ml) % Positive Probability 45 100 5.715 100 (4.8 to 7) 5 90 1.67 45 0.56 15 0 0

The results shown in Table 12 clearly indicated that HBV nucleic acidswere detected in the multiplex reaction that was also capable ofdetecting HIV-1 and HCV nucleic acids. Moreover, the nucleic acids ofHBV genotypes A-G were all detectable in this assay. As indicatedhereinabove, the collection of HBV specific oligonucleotides used inthis procedure can be used in stand-alone assays for detecting HBV, orin combination with the oligonucleotides specific for HIV, oralternatively HCV, to create duplex assays.

To further illustrate the versatility of the above-described analytedetection systems, amplicon production was monitored as a function oftime in “real-time” amplification procedures. Amplicon-specificmolecular beacons that were included in the amplification reactionsprovided a means for continuous monitoring of amplicon synthesis.Fluorescent emissions that increased with time indicated the productionof amplicons that hybridized to the molecular beacon and caused adetectable transition to the “open” conformation of the probe.

Molecular beacons comprise nucleic acid molecules having atarget-complementary loop sequence, an affinity pair (or nucleic acid“arms”) that interact to form a “stem” structure by complementary basepairing in the absence of a target (i.e., the “closed” conformation),and a paired set of labels that interact when the probe is in the closedconformation. Hybridization of the target nucleic acid and thetarget-complementary sequence of the probe causes the members of theaffinity pair to separate, thereby shifting the probe to the openconfirmation. This shift is detectable by virtue of reduced interactionbetween the members of label pair, which may be, for example, afluorophore and a quencher. Molecular beacons are fully described inU.S. Pat. No. 5,925,517, the disclosure of this patent document beingincorporated by reference herein.

Commercially available software was used to analyze time-dependentresults obtained using molecular beacons that were specific for HBV,HIV-1 or HCV amplicons. Results from these analyses indicated a stronglinear relationship between the number of target copies included in anamplification reaction and the time at which the fluorescent signalexceeded a background threshold (i.e., “time-of-emergence” abovebackground). As confirmed by the results presented below, theseprocedures were useful for quantifying analyte target amounts over avery broad range. More particularly, when known amounts of analytepolynucleotides are used as calibration standards, it is possible todetermine the amount of analyte present in a test sample by comparingthe measured time-of-emergence with the standard plot.

The fact that the amplification reaction used in the below-describedprocedures operated at constant temperature and without interruption fora separate detection step, so that amplification and detection tookplace simultaneously, imposed strict requirements on the molecularbeacons. More specifically, success in the procedure required that themolecular beacon bind amplicon without inhibiting subsequent use of theamplicon as a template in the exponential amplification mechanism.Indeed, the finding that an amplification reaction could proceedefficiently in the presence of a molecular beacon indicated thatinteraction of the probe with its target did not irreversibly inhibit orpoison the amplification reaction.

Example 10 describes procedures wherein molecular beacon probes, eachlabeled with an interactive fluorophore/quencher pair, were used formonitoring time-dependent amplicon production in TMA reactions. Althoughthe molecular beacons described in this Example hybridized to only onestrand of the amplified nucleic acid product, complementary probesequences also would be expected to hybridize to the opposite nucleicacid strand, and so fall within the scope of the invention.

EXAMPLE 10 Real-Time Monitoring of Amplicon Production

Molecular beacons having binding specificity for HBV, HIV-1 or HCVamplicons were synthesized by standard solid-phase phosphite triesterchemistry using 3′ quencher-linked controlled pore glass (CPG) and 5′fluorophore-labeled phosphoramidite on a Perkin-Elmer (Foster City,Calif.) EXPEDITE model 8909 automated synthesizer. Fluorophores used forconstructing the molecular beacons included fluorescein, ROX and CY5dyes. BLACK HOLE QUENCHER 2 (Biosearch Technologies, Inc.; Novato,Calif.) and DABCYL were used as quenchers. All of the molecular beaconswere constructed using 2′-methoxy nucleotide analogs. The CPG andphosphoramidite reagents were purchased from Glen Research Corporation(Sterling, Va.). Following synthesis, the probes were deprotected andcleaved from the solid support matrix by treatment with concentratedammonium hydroxide (30%) for two hours at Wt. Next, the probes werepurified using polyacrylamide gel electrophoresis followed by HPLC usingstandard procedures that will be familiar to those having an ordinarylevel of skill in the art.

The HBV, HIV-1 and HCV nucleic acid targets used in the procedure wereartificial or synthetic targets of known concentration. The HBV targetwas a single-stranded M13 clone containing a portion of the HEY genomethat included sequences corresponding to, or complementary to each ofthe primers. The HIV-1 target was a synthetic transcript. The HCV targetwas an ARMORED RNA (Ambion, Inc.; Austin, Tex.) that included HCVgenomic sequences. ARMORED RNA® technology is used for producingribonuclease-resistant RNA controls and standards by assembling specificRNA sequences and viral coat proteins into pseudo-viral particles.Molecular beacons were used at a level of about 1.5 pmoles/reaction.Reactions for amplifying HBV contained 5×10²−5×10⁹ templatecopies/reaction. Reactions for amplifying HIV-1 contained 5×10¹−5×10⁶template copies/reaction. Reactions for amplifying HCV contained 5−5×10⁸template copies/reaction.

Tubes containing 15 μl of a buffered solution that included salts andreagents essentially as described under Example 2, a targetpolynucleotide, and a molecular beacon were first overlaid with 15 μl ofinert oil to prevent evaporation. The tubes were then incubated in a dryheat block for 10 minutes at 60° C. to facilitate primer annealing.Primers for amplifying the HBV target had the target-complementarysequences of SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:15 and SEQ ID NO:11.Primers for amplifying the HIV-1 target had the target-complementarysequences of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:103 and SEQ IDNO:102. Primers for amplifying the HCV target had thetarget-complementary sequences of SEQ ID NO:115, SEQ ID NO:117 and SEQID NO:118. Notably, amplification reactions used for testing theHBV-specific molecular beacons included all of the primers foramplifying all three targets. Amplification reactions used for testingthe HIV-1 and HCV-specific molecular beacons included all of the primersfor amplifying both of these targets, but not primers for amplifying theHBV target. Of course, it is intended that any of the below-describedmolecular beacons could be used in amplification reactions that includeany combination of primers. Following the 60° C. incubation step, tubeswere transferred to a 42° C. heat block and then incubated for 10minutes. Five microliter aliquots of an enzyme reagent that includedboth MMLV reverse transcriptase and T7 RNA polymerase enzymes were addedto each of the tubes using a multichannel pipettor. Tubes were vortexedbriefly and then transferred to a ROTORGENE-2000 (Corbett Research;Sydney, Australia) rotor that had been pre-warmed to 42° C.Amplification reactions were carried out at 42° C., fluorescencereadings were taken every 30 seconds, and the results analyzed inreal-time using standard software that was bundled with theROTORGENE-2000 instrument.

Table 13 presents the target-complementary loop sequences ofHBV-specific molecular beacons that were used during the development ofthe invention. Each of the molecular beacons included an appended 5′CCGAG arm sequence, and an appended 3′ CUCGG arm sequence. Each probeincluded a CY5 fluorophore at its 5′-end, and a BLACK HOLE QUENCHER 2moiety at its 3′-end. All of the HBV-specific molecular beacons hadtarget-complementary loop sequences that were 12-20 nucleotides inlength and included 12-20 contiguous nucleotides contained within thesequence AAGAAGATGAGGCATAGCAGCAGGATGAAGAGGAA SEQ ID NO:126, allowing forthe presence of nucleotide analogs and RNA and DNA equivalents.

TABLE 13 Target-Complementary Loop Sequences ofHBV-Specific Molecular Beacons Sequence (5′ to 3′) IdentifierGAAGAUGAGGCAUAGCAG SEQ ID NO: 127 AAGAAGAUGAGGCAUAGCAG SEQ ID NO: 128CAGCAGGAUGAAGAGGAA SEQ ID NO: 129 CAGGAUGAAGAGGA SEQ ID NO: 130AAGAAGAUGAGG SEQ ID NO: 131 GAAGAUGAGGCAUAGC SEQ ID NO: 132GAAGAUGAGGCAUA SEQ ID NO: 133

The results presented in Table 14 confirmed that amplification reactionswhich included one of the HBV-specific molecular beacons desirablyproduced a fluorescent signal that increased with time. All results werebased on reactions that were conducted in triplicate. Significantly, thedifferent molecular beacons behaved somewhat differently in thereal-time assay format. For example, reactions that included the highlypreferred molecular beacon having the target-complementary loop sequenceof SEQ ID NO:127 gave rapid detection of high target numbers and astrong linear relationship between the fluorescent signal and targetamount on a logarithmic plot over the full range of input target levelstested (see FIG. 3). Coefficients of variation (CVs) for thetime-of-emergence readings obtained using this probe were 3.2% or less,thereby indicating very high levels of precision among the data points.Reactions that included the molecular beacon having thetarget-complementary loop sequence of SEQ ID NO:133 exhibited differentresponse characteristics that were somewhat less linear over the rangeof target input levels tested due to the probe response at low targetlevels. This latter probe will be particularly useful for detecting andquantifying HBV target amounts greater than 5,000 copies of HBV.

TABLE 14 Measured Time-of-Emergence for Different Molecular BeaconsTime-of-Emergence for Different Molecular Beacons (minutes) HBV TargetSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID copies/rxn NO: 127 NO:128 NO: 129 NO: 130 NO: 131 NO: 132 NO: 133 5 × 10⁹ 4.4 NT NT 3.3 8.9 NTNT 5 × 10⁸ 6.7 NT NT 5.4** 14.9 NT NT 5 × 10⁷ 8.7 NT NT 6.2* 23.4 10.110.0 5 × 10⁶ 11.1 NT NT NT NT 12.3 12.2 5 × 10⁵ 13.4 16.3 20.9  NT NT14.5 13.9 5 × 10⁴ 15.9 18.6 33.5* NT NT 17.2 17.6 5 × 10³ 18.3  29.7* NDNT NT  21.9* 21.5* 5 × 10² 20.8** NT NT NT NT ND 49.7* “NT” = not tested“ND” = not detected *= only one out of three replicates reactive **=only two out of three replicates reactive

Table 15 presents the target-complementary loop sequences ofHIV-specific molecular beacons that were used during the development ofthe invention. The molecular beacon having the target-complementary loopsequence of SEQ ID NO:134 included an appended 5′ CCGAG arm sequence,and an appended 3′ CUCGG arm sequence. This probe included a ROXfluorophore at its 5′-end, and a BLACK HOLE QUENCHER 2 quencher moietyat its 3′-end. A related molecular beacon had an identicaltarget-complementary loop sequence, but had the overall sequence5′-CCGAGAGGGUACAGUGCAGGGGUCUCGG-3′ (SEQ ID NO:135). When compared withthe above-described HIV-specific molecular beacon, the arms of thisprobe each included a single additional nucleotide immediately adjacentto the target-complementary loop sequence of the probe. This probe alsoincluded a fluorescein fluorophore (instead of ROX) at its 5′-end, and aDABCYL quencher moiety (instead of the BLACK HOLE 2 QUENCHER 2) at its3′-end. The molecular beacon having the target-complementary loopsequence of SEQ ID NO:136 included an appended 5′ CCGAGA arm sequence,and an appended 3′ UCUCGG arm sequence. This probe also included afluorescein fluorophore at its 5′-end, and DABCYL quencher moiety at its3′-end. The molecular beacons used in these procedures hadtarget-complementary loop sequences that were 16-17 nucleotides inlength, and included 16-17 contiguous nucleotides contained within thesequence GGGGTACAGTGCAGGGG (SEQ ID NO:137), allowing for the presence ofnucleotide analogs and RNA and DNA equivalents.

TABLE 15 Target-Complementary Loop Sequences ofHIV-Specific Molecular Beacons Sequence (5′ to 3′) IdentifierGGGUACAGUGCAGGGG SEQ ID NO: 134 AGGGGUACAGUGCAGGGGU SEQ ID NO: 136

The results presented in Table 16 confirmed that amplification reactionswhich included one of the HIV-specific molecular beacons desirablyproduced a fluorescent signal that increased with time. The molecularbeacon that included the target-complementary loop sequence of SEQ IDNO:134 allowed rapid detection of low target levels. Reactions thatincluded this probe were conducted in triplicate and exhibited a stronglinear relationship between input target level and fluorescent signalwith at least four logs of useful dynamic range extending from5×10¹−5×10⁵ copies/reaction of the HIV-1 target. Although the resultsare not presented in the table, additional testing showed that thedynamic range of reactions that included this molecular beacon extendedup to 5×10⁷ or 5×10⁸ copies/reaction of the HIV-1 target. The molecularbeacon having the sequence of SEQ ID NO:135 exhibited a biphasic plot ona logarithmic graph of input target amount against time-of-emergence.These reactions, which were conducted in replicates of five, exhibited astrong linear relationship between input target amount andtime-of-emergence in the range of 5×10³−5×10⁶, and a somewhat weakerlinear relationship in the range of 5×10¹−5×10³ copies/reaction of theHIV-1 target. This molecular beacon is particularly useful forquantifying the higher range of target amounts in real-time assays, butexhibited a useful dynamic range of at least five logs that covered therange extending from 5×10¹−5×10⁶ copies/reaction of the HIV-1 template.Reactions that included the molecular beacon having thetarget-complementary loop sequence of SEQ ID NO:136 were conducted intriplicate and exhibited a strong linear relationship between inputtarget level and fluorescent signal with at least three logs of usefuldynamic range that extended from 5×10²−5×10⁵ copies/reaction of theHIV-1 target. Interestingly, amplification reactions that included thismolecular beacon required slightly more time for detecting low targetamounts when compared with reactions that included either of the othertwo HIV-specific molecular beacons.

TABLE 16 Measured Time-of-Emergence for Different Molecular BeaconsTime-of-Emergence for Different HBV Target Molecular Beacons (minutes)(copies/rxn) SEQ ID NO: 134 SEQ ID NO: 135 SEQ ID NO: 136 5 × 10⁶ NT 4.0NT 5 × 10⁵ 7.2 5.8 6.1 5 × 10⁴ 8.8 8.0 9.3 5 × 10³ 10.6 9.7 11.2 5 × 10²12.7 12.7 14.2 5 × 10¹ 14.8 17.0 ND “NT” = not tested “ND” = notdetected

Table 17 presents the target-complementary loop sequences ofHCV-specific molecular beacons that were used during the development ofthe invention. The molecular beacon that included thetarget-complementary loop sequence of SEQ ID NO:138 included an appended5′ CGAGG arm sequence, and an appended 3′ CCUCG arm sequence. Thismolecular beacon additionally included a CY5 fluorophore at its 5′-end,and a BLACK HOLE QUENCHER 2 moiety at its 3′-end. The molecular beaconthat included the target-complementary loop sequence of SEQ ID NO:139included an appended 5′ CCGAG arm sequence, and an appended 3′ CUCGG armsequence. This molecular beacon was labeled with a fluoresceinfluorophore at its 5′-end, and with a DABCYL moiety at its 3′-end. Themolecular beacon that included the target-complementary loop sequence ofSEQ ID NO:140 included an appended 5′ CGGAC arm sequence, and anappended 3′ GUCCG arm sequence. This molecular beacon was labeled at its5′-end with a CY5 fluorophore, and at its 3′-end with a BLACK HOLEQUENCHER 2 moiety. The molecular beacons harboring thetarget-complementary loop sequences of the probes that included thetarget-complementary loop sequences of SEQ ID NO:138, SEQ ID NO:139 andSEQ ID NO:140 were tested in replicates of five, three and four,respectively. All of the molecular beacons used in these procedures hadtarget-complementary loop sequences that were 10-14 nucleotides inlength and included 10-14 contiguous nucleotides contained within thesequence TAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGC (SEQ ID NO:141), allowing for the presenceof nucleotide analogs and RNA and DNA equivalents.

TABLE 17 Target-Complementary Loop Sequences ofHCV-Specific Molecular Beacons Sequence (5′ to 3′) Identifier AACCGGUGAGSEQ ID NO: 138 UACACCGGAAUUGC SEQ ID NO: 139 UAGUAUGAGUGUCSEQ ID NO: 140

The results presented in Table 18 confirmed that amplification reactionswhich included one of the HCV-specific molecular beacons desirablyproduced a fluorescent signal that increased with time. Reactions thatincluded the molecular beacon having the target-complementary loopsequence of SEQ ID NO:138 exhibited a useful dynamic range of about sixlogs, with good results being achieved in the range of target amountsextending from 5×10¹−5×10¹ copies/reaction of the HCV template.Reactions that included the molecular beacon having thetarget-complementary loop sequence of SEQ ID NO:139 exhibited a usefuldynamic range of about seven logs, with good results being achieved inthe range of about 5×10¹−5×10⁸ copies/reaction of the HCV template.Reactions that included the molecular beacon having thetarget-complementary loop sequence of SEQ ID NO:140 exhibited a usefuldynamic range of about eight logs, with excellent results being achievedover the full range of about 5−5×10⁸ copies/reaction of the HCVtemplate.

TABLE 18 Measured Time-of-Emergence for Different Molecular BeaconsTime-of-Emergence for Different HCV Target Molecular Beacons (minutes)(copies/rxn) SEQ ID NO: 138 SEQ ID NO: 139 SEQ ID NO: 140 5 × 10⁸ NT 1.02.3 5 × 10⁷ 6.9 3.3 6.5 5 × 10⁶ 8.3 5.3 8.0 5 × 10⁵ 10.4 7.5 10.1 5 ×10⁴ 12.2 9.2 11.8 5 × 10³ 14.0 11.2 14.3 5 × 10² 16.1 14.2 16.0 5 × 10¹18.5 20.0 18.3 5 NT 27.4* 21.0** “NT” = not tested *= only one out ofthree replicates reactive **= only three out of four replicates reactive

This invention has been described with reference to a number of specificexamples and embodiments thereof. Of course, a number of differentembodiments of the present invention will suggest themselves to thosehaving ordinary skill in the art upon review of the foregoing detaileddescription. Thus, the true scope of the present invention is to bedetermined upon reference to the appended claims.

What is claimed is:
 1. A composition of oligonucleotides for amplifyingand detecting an HBV nucleic acid wherein said composition comprises atleast two amplification oligomers configured to generate an HBV ampliconfrom an HBV nucleic acid present in a test sample at a concentration aslow as 200 copies/ml of reaction mixture, wherein: (a) the firstamplification oligomer consists of a target hybridizing sequence,optionally, an upstream sequence that is not complementary to an HBVtarget sequence, and a 5′ promoter sequence for an RNA polymerase,wherein the target hybridizing sequence consists of SEQ ID NO:25 or SEQID NO:28; and (b) the second amplification oligomer consists of a targethybridizing sequence consisting of SEQ ID NO:15.
 2. The composition ofclaim 1, wherein the amplification oligomer at (a) is selected from thegroup consisting of: SEQ ID NOS:42, 45, and combinations thereof.
 3. Thecomposition of claim 1, wherein the composition is provided as acomponent of a kit for the detection of HBV nucleic acid.
 4. Thecomposition of claim 1, wherein the composition is provided as acomponent of a kit for screening donated blood samples or plasma samplesfor the presence of HBV.
 5. The composition of claim 4, furthercomprising components for amplification and/or detection of HIV-1nucleicacids, HCV nucleic acids or both.
 6. The composition of claim 1, furthercomprising a detectably labeled probe complementary to an HBV amplicon.7. The composition of claim 6, wherein the detectably labeled probecomprises a fluorescent label.
 8. The composition of claim 6, whereinsaid detectably labeled probe is a molecular beacon.
 9. A method forscreening donated blood or blood products for the presence of HBV,comprising the steps of: a. contacting a test sample with (i) anamplification oligomer, consisting of a target hybridizing sequence,optionally, an upstream sequence that is not complementary to an HBVtarget sequence, and a 5′ promoter sequence for an RNA polymerase,wherein the target hybridizing sequence consists of SEQ ID NO:25 or SEQID NO:28; and (ii) an amplification oligomer consisting of a targethybridizing sequence consisting of SEQ ID NO:15; and b. amplifying anyHBV nucleic acid that is present in the test sample in an isothermal invitro nucleic acid amplification reaction to generate an HBV amplicon;and c. detecting the HBV amplicon; wherein detecting the presence of HBVin the test sample indicates that the blood product or the donated bloodcontains HBV nucleic acids, and wherein said method is capable ofdetecting HBV nucleic acid at a concentration at least as low as 200copies/ml of reaction mixture.
 10. The method of claim 9, furthercomprising a separating step to separate HBV nucleic acids from one ormore other components in a biological sample.
 11. The method of claim10, wherein the separating step is performed using a target captureoligomer that includes a first sequence complementary to an HBV sequenceand includes a second sequence for immobilization to a solid support.12. The method of claim 9, wherein the amplification oligomer at a.(i)consists of deoxyribonucleic acid residues, or the amplificationoligomer at a.(ii) consists of deoxyribonucleic acid residues, or acombination thereof.
 13. The method of claim 9, wherein the isothermalin vitro amplification is a real-time amplification.
 14. The method ofclaim 9, wherein steps b. and c. are performed simultaneously.
 15. Themethod of claim 9, wherein the amplification oligomer at a.(i) isselected from the group consisting of: SEQ ID NOS:42, 45, andcombinations thereof.
 16. The method of claim 9, wherein step c. isperformed using a detection probe oligomer.
 17. The method of claim 16,wherein the detection probe oligomer is labeled with a chemiluminescentcompound, a fluorescent compound, a quencher compound or a combinationthereof.
 18. The method of claim 9, wherein the test sample is derivedfrom a pool of blood donated by two or more blood donors.
 19. The methodof claim 9, wherein the isothermal in vitro nucleic acid amplificationreaction is a multiplex nucleic acid amplification reaction thatamplifies HIV-1nucleic acid, HCV nucleic acid, or HIV-1nucleic acid andHCV nucleic acid in addition to HBV nucleic acid.
 20. The method ofclaim 9, wherein the detecting step comprises detecting HBV genotype Gamplicons.
 21. A method for screening plasma for the presence of HBV,comprising the steps of: a. contacting a test sample with (i) anamplification oligomer consisting of a target hybridizing sequence,optionally, an upstream sequence that is not complementary to an HBVtarget sequence, and a 5′ promoter sequence for an RNA polymerase,wherein the target hybridizing sequence consists of SEQ ID NO:25 or SEQID NO:28; and (ii) an amplification oligomer consisting of a targethybridizing sequence consisting of SEQ ID NO:15; and b. amplifying anyHBV nucleic acid that is present in the test sample in an isothermal invitro nucleic acid amplification reaction to generate an HBV amplicon;and c. detecting the HBV amplicon; wherein detecting the presence of HBVin the test sample indicates that the plasma contains HBV nucleic acids,and wherein said method is capable of detecting HBV nucleic acid at aconcentration at least as low as 200 copies/ml of reaction mixture. 22.The method of claim 21, further comprising a separating step to separateHBV nucleic acids from one or more other components in a biologicalsample.
 23. The method of claim 22, wherein the separating step isperformed using a target capture oligomer that includes a first sequencecomplementary to an HBV sequence and includes a second sequence forimmobilization to a solid support.
 24. The method of claim 21, whereinthe amplification oligomer at a.(i) consists of deoxyribonucleic acidresidues, or the amplification oligomer at a.(ii) consists ofdeoxyribonucleic acid residues, or a combination thereof.
 25. The methodof claim 21, wherein the isothermal in vitro amplification is areal-time amplification.
 26. The method of claim 21, wherein steps b.and c. are performed simultaneously.
 27. The method of claim 21, whereinthe amplification oligomer at a.(i) is selected from the groupconsisting of: SEQ ID NOS: 42, 45, and combinations thereof.
 28. Themethod of claim 21, wherein step c. is performed using a detection probeoligomer.
 29. The method of claim 28, wherein the detection probeoligomer is labeled with a chemiluminescent compound, a fluorescentcompound, a quencher compound or a combination thereof.
 30. The methodof claim 21, wherein the test sample is further tested for the presenceof HIV-1nucleic acid, HCV nucleic acid or both.
 31. The method of claim21, wherein the isothermal in vitro nucleic acid amplification reactionis a multiplex nucleic acid amplification reaction that amplifiesHIV-1nucleic acid, HCV nucleic acid, or HIV-1nucleic acid and HCVnucleic acid in addition to HBV nucleic acid.
 32. The method of claim21, wherein the detecting step comprises detecting HBV genotype Gamplicons.
 33. The method of claim 21, wherein the test sample isderived from a pool of plasma donated by two or more donors.